202192Orig1s000 - Food and Drug Administration · 2012. 1. 9. · NDA # 202,192 Reviewer: Wei Chen, Ph.D. 5 following a single oral dose of INCB018424. Administration of ruxolitinib

CENTER FOR DRUG EVALUATION AND RESEARCH

APPLICATION NUMBER:

202192Orig1s000

PHARMACOLOGY REVIEW(S)

MEMORANDUM ruxolitinib (JAKAFI) Date: October 27, 2011 To: File for NDA 202192 From: John K. Leighton, PhD, DABT Director, Division of Hematology Oncology Toxicology Office of Hematology and Oncology Products I have examined pharmacology/toxicology supporting review of Dr. Chen and labeling and supervisory memorandum provided by Dr. Saber. I concur with Dr Saber’s conclusion that JAKAFI may be approved for the proposed indication and that no additional nonclinical studies are needed.

Reference ID: 3035562

---------------------------------------------------------------------------------------------------------This is a representation of an electronic record that was signedelectronically and this page is the manifestation of the electronicsignature.---------------------------------------------------------------------------------------------------------/s/----------------------------------------------------

JOHN K LEIGHTON10/27/2011


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MEMORANDUM Date: October 27, 2011 From: Haleh Saber, Ph.D. Pharmacology/Toxicology Supervisor

Division of Hematology Oncology Toxicology (DHOT) Office of Hematology Oncology Products (OHOP)

Re: Approvability for Pharmacology and Toxicology NDA: 202,192 Drug: JAKAFI (ruxolitinib) Indication: For treatment of patients with myelofibrosis, including primary

myelofibrosis, post-polycythemia vera myelofibrosis and post-essential thrombocythemia myelofibrosis

Ruxolitinib is a small molecule inhibitor of Janus Associated Kinases 1 and 2 (JAK 1/2). The inhibitory activity of ruxolitinib on JAKs was demonstrated in vitro and in animal models containing aberrant JAK/STAT signaling. Myelofibrosis, a type of myeloproliferative disorder, is known to be associated with dysregulation of JAK 1/2. This condition may present as a primary myelofibrosis or it may be secondary to polycythemia vera or essential thrombocythemia. The pharmacologic class assigned to ruxolitinib is “kinase inhibitor”, as presented in the HIGHLIGHTS section of the label. The detailed information on the mechanism of action of the drug is under section 12.1 of the FULL PRESCRIBING INFORMATION. Pharmacology, safety pharmacology, pharmacokinetic/ADME (absorption, distribution, metabolism and excretion), and toxicology studies were conducted in in vitro systems and/or in animal species. Ruxolitinib was administered orally to animals in toxicology studies, consistent with the intended route of administration in patients. Drug-related toxicities were similar after single- or repeat-dose administration, therefore only repeat-dose general toxicology studies were reviewed for this NDA. Toxicities were mostly related to pharmacology of the drug, with lymphoid depletion, and reduced size of thymus and spleen being the primary adverse effects. Single-dose administration of ruxolitinib to Beagle dogs in a safety pharmacology study resulted in reduced systolic, diastolic, and mean arterial pressure. Safety pharmacology studies also revealed CNS and respiratory effects in animals; e.g. lower body temperature, reduced activity, and increased tidal volume/decreased minute volume. While dizziness and balance disorder have been reported in patients treated with ruxolitinib, cardiovascular or respiratory adverse effects have not been observed. When administered during the period of organogenesis, ruxolitinib was not teratogenic to rats or rabbits. Reduced fetal weight and/or increased post-implantation loss were seen in animals only at doses that resulted in maternal mortalities. In a designated fertility study, ruxolitinib did not impair male or female fertility but resulted in increased post-implantation loss. In a peri- and post-natal developmental study conducted in rats, there were no drug-related adverse findings in pups for fertility indices or for maternal or


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embryofetal survival, growth, and development parameters at the doses evaluated. Reduced number of pups (F1) delivered compared to the control appears to be secondary to the post-implantation loss, as previously reported in the embryo-fetal developmental study. Ruxolitinib was not genotoxic when tested in vitro or in vivo for mutagenic or clastogenic potential. When tested in a 6-motnh carcinogenicity study in Tg.rasH2 transgenic mouse, ruxolitinib was not carcinogenic. A 2-year carcinogenicity study in rat is ongoing. Considering the serious and life-threatening condition of the disease and lack of adequate therapy, the results of the 2-year carcinogenicity study may be submitted post-approval. Based on the QSAR (quantitative structure–activity relationship) models, six impurities were found to have structural alerts for genotoxicity. Further evaluation by Ames assay, showed these compounds to be negative for mutagenic potential. The Ames tests on impurities were non-GLP; however, upon further examination of the study criteria, conduct of the study, and concentrations used in the study, DHOT accepts results of the non-GLP studies for these impurity. Of note, one of the six impurities was present in the 6-month carcinogenicity study at Considering the results of reproduction toxicology studies together with the negative results reported in the genetic toxicology studies, the Applicant’s proposed Category C for pregnancy is acceptable. The nonclinical studies were reviewed by Dr. Wei Chen. Results of the carcinogenicity study were reviewed by Dr. Miyun Tsai-Turton. The nonclinical findings are summarized in the “Executive Summary” of the NDA review and reflected in the product label. Recommendation: I concur with Dr. Chen that from a nonclinical perspective, JAKAFI may be approved for the proposed indication.


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HALEH SABER10/27/2011


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DEPARTMENT OF HEALTH AND HUMAN SERVICES PUBLIC HEALTH SERVICE

FOOD AND DRUG ADMINISTRATION CENTER FOR DRUG EVALUATION AND RESEARCH

PHARMACOLOGY/TOXICOLOGY NDA/BLA REVIEW AND EVALUATION

Application number: 202,192

Supporting document/s: SDN 000

Applicant’s letter date: June 3, 2011

CDER stamp date: June 3, 2011

Product: Ruxolitinib phosphate

Indication: Myelofibrosis

Applicant: Incyte Corporation

Review Division: Division of Hematology Oncology Toxicology

(for Division of Hematology Products; DHP)

Reviewer: Wei Chen, Ph.D.

Supervisor/Team Leader: Haleh Saber, Ph.D.

Division Director: John Leighton, Ph.D., D.A.B.T.

(Ann Farrell, MD for DHP)

Project Manager: Amy Baird

Disclaimer Except as specifically identified, all data and information discussed below and necessary for approval of NDA 202,192 are owned by Incyte Corporation or are data for Incyte Corporation has obtained a written right of reference. Any information or data necessary for approval of NDA 202,192 that Incyte Corporation does not own or have a written right to reference constitutes one of the following: (1) published literature, or (2) a prior FDA finding of safety or effectiveness for a listed drug, as reflected in the drug’s approved labeling. Any data or information described or referenced below from reviews or publicly available summaries of a previously approved application is for descriptive purposes only and is not relied upon for approval of NDA 202,192.


NDA # 202,192 Reviewer: Wei Chen, Ph.D.

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TABLE OF CONTENTS 1 EXECUTIVE SUMMARY ......................................................................................... 4

1.1 INTRODUCTION.................................................................................................... 4 1.2 BRIEF DISCUSSION OF NONCLINICAL FINDINGS ...................................................... 4 1.3 RECOMMENDATIONS............................................................................................ 5

2 DRUG INFORMATION ............................................................................................ 6

2.1 DRUG ................................................................................................................. 6 2.2 RELEVANT INDS, NDAS, BLAS AND DMFS........................................................... 6 2.3 DRUG FORMULATION ........................................................................................... 6 2.4 COMMENTS ON NOVEL EXCIPIENTS....................................................................... 7 2.5 COMMENTS ON IMPURITIES/DEGRADANTS OF CONCERN ......................................... 7 2.6 PROPOSED CLINICAL POPULATION AND DOSING REGIMEN ...................................... 7

3 STUDIES SUBMITTED............................................................................................ 8

3.1 STUDIES REVIEWED............................................................................................. 8 3.2 STUDIES NOT REVIEWED ................................................................................... 11 3.3 PREVIOUS REVIEWS REFERENCED...................................................................... 13

4 PHARMACOLOGY................................................................................................ 15

4.1 PRIMARY PHARMACOLOGY................................................................................. 15 4.2 SECONDARY PHARMACOLOGY............................................................................ 22 4.3 SAFETY PHARMACOLOGY................................................................................... 23

5 PHARMACOKINETICS/ADME/TOXICOKINETICS .............................................. 27

5.1 PK/ADME........................................................................................................ 27

6 GENERAL TOXICOLOGY................................................................................... 110

6.1 SINGLE-DOSE TOXICITY ................................................................................... 110 6.2 REPEAT-DOSE TOXICITY .................................................................................. 111

7 GENETIC TOXICOLOGY .................................................................................... 138

7.1 IN VITRO REVERSE MUTATION ASSAY IN BACTERIAL CELLS (AMES)..................... 138 7.2 IN VITRO ASSAYS IN MAMMALIAN CELLS............................................................ 140 7.3 IN VIVO CLASTOGENICITY ASSAY IN RODENT (MICRONUCLEUS ASSAY)................ 142

8 CARCINOGENICITY ........................................................................................... 144

9 REPRODUCTIVE AND DEVELOPMENTAL TOXICOLOGY .............................. 154

9.1 FERTILITY AND EARLY EMBRYONIC DEVELOPMENT............................................. 154 9.2 EMBRYONIC FETAL DEVELOPMENT ................................................................... 157 9.3 PRENATAL AND POSTNATAL DEVELOPMENT....................................................... 166

10 SPECIAL TOXICOLOGY STUDIES................................................................. 169 11 OTHER TOXICOLOGY STUDIES 169



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12 INTEGRATED SUMMARY AND SAFETY EVALUATION............................... 171

13 APPENDIX/ATTACHMENTS........................................................................... 177



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1 Executive Summary

1.1 Introduction Ruxolitinib phosphate, a new molecular entity, is a small molecule inhibitor of the

Janus kinase family of protein tyrosine kinases (JAKs). JAKs play a role in the signal transduction following cytokine and growth factor binding to their receptors. After the receptor associates with its respective cytokine/ligand it goes through a conformational change, bringing the JAKs close enough to phosphorylate each other. Once phosphorylated/ activated, the JAKs transduce an intracellular signal by promoting and activating transcription factors called STATs. The activated STATs (signal transducer and activator of transcription) dissociate from the receptor and form dimers before translocating to the cell nucleus where they regulate transcription of selected genes [Kisseleva et al, Gene 285:1-24,(2002)]. The efficacy of ruxolitinib phosphate has been shown in in vitro studies and in in vivo nonclinical tumor models representing both hematological and solid tumors. Ruxolitinib is being developed as a monophosphate salt for the oral treatment of myelofibrosis, a type of myeloproliferative neoplasm (MPN). With this NDA, the Applicant is submitting nonclinical pharmacology, pharmacokinetic, and toxicology studies to support the approval of ruxolitinib phosphate for the treatment of myelofibrosis.

1.2 Brief Discussion of Nonclinical Findings Ruxolitinib was rapidly absorbed in rats and dogs with a similarly short half-life after single and multiple oral dosing. The pivotal non-clinical general toxicity studies of ruxolitinib were conducted in rats and dogs, consistent with the clinical route of administration. Non-clinical studies also included safety pharmacology studies, reproductive toxicity studies, genotoxicity studies, and a carcinogenicity study. The pivotal toxicology studies were conducted in compliance with Good Laboratory Practice regulation. Nonclinical pharmacokinetic and toxicokinetic studies of ruxolitinib were also evaluated in rats and dogs. Pharmacology Ruxolitinib (also referred to as “INCB018424” or “INC424”) is a pyrrolo-pyrimidine derivative, and it is an inhibitor of JAKs, with relative selectivity for JAK1 and JAK2. Ruxolitinib inhibited JAK in enzyme-based and cell-based assays. Ruxolitinib showed its inhibitory activity on JAKs in animal models with aberrant JAK/STAT signaling. Ruxolitinib inhibited cell proliferation, decreased circulating inflammatory cytokines (eg, TNF-α, IL-6) and resulted in prolonged survival in the mice xenograft models of hematological malignancies. Safety pharmacology Safety pharmacology studies revealed adverse effects of ruxolitinib on the functions of central nervous, respiratory and cardiovascular systems. Administration of ruxolitinib resulted in various observations including lower body temperature, lower activity and observations of darkened skin and mucous membranes. Decreased respiratory frequency, increase in tidal volume and decrease in minute volume were noted in rats



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following a single oral dose of INCB018424. Administration of ruxolitinib at a dose of 30 mg/kg (the highest dose tested) resulted in significantly lower pulse pressure, as well as lower systolic, diastolic, and calculated mean arterial pressure (up to 53%, 41%, 31%, and 33%, respectively) when compared to the control group. General toxicology The repeated dose toxicology studies in rats and dogs have shown that oral administration of ruxolitinib resulted in alterations in hematology parameters and microscopic observations of marked lymphoid depletion, which correlated with small thymus and spleen as well as low lymphocyte counts. No adverse effect of ruxolitinib on central nervous system, respiratory, or cardiovascular system was noted at the doses tested in the repeat-dose toxicology studies in rats and dogs. Clinical and anatomical pathology findings associated with the treatment of ruxolitinib were generally reversible after a recovery period. Toxicities observed are mostly direct effects based on the pharmacology of ruxolitinib. Genetic toxicology Ruxolitinib was not mutagenic or clastogenic in the in vitro and in vivo assays studied. Reproductive toxicology Ruxolitinib did not impair fertility when administered to either male or female rats prior to and during the mating time frame. However, embryo-fetal viability was reduced. Ruxolitinib was not teratogenic in either the rat or the rabbit when administered to animals during the period of organogenesis. It did, however, lead to increased postimplantation loss and/or reduced fetal weight at a maternally toxic/lethal dose. In a pre- and post-natal developmental study, ruxolitinib resulted in a slightly prolonged gestation period; the significance of this finding is unclear. Reduced number of pups (F1) delivered appears to be secondary to the increased post-implantation loss in F0 females.. Carcinogenicity studies Treatment of Tg.rasH2 animals with ruxolitinib at daily oral doses up to 125 mg/kg for 26-weeks did not increase the incidence of neoplastic lesions.

1.3 Recommendations 1.3.1 Approvability

Recommending approval. The non-clinical studies adequately support the safety of ruxolitinib phosphate by oral route in myelofibrosis.

1.3.2 Additional Non Clinical Recommendations

Additional non-clinical studies are not needed at this time. 1.3.3 Labeling Information needed for nonclinical sections of the label are provided in this review. Therefore, a separate labeling review is not deemed necessary.



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2 Drug Information

2.1 Drug CAS Registry Number: 941678-49-5 Generic Name: ruxolitinib phosphate Code Name: INCB018424; INC424 Chemical Name: (R)-3-(4-(7H-pyrrolo [2,3-d] pyrimidin-4-yl)-1H-pyrazol-1-yl)-3-cyclopentylpropanenitrile phosphate Molecular Formula/Molecular Weight: C17H21N6O4P / 404.36 Structure or Biochemical Description

Pharmacologic Class: kinase inhibitor Mechanism of action: inhibition of the Janus Associated Kinases 1 and 2 (JAKs 1 and 2) 2.2 Relevant INDs, NDAs, BLAs and DMFs: IND 77456 (for myeloproliferative disorders) IND 77101 (for psoriasis) IND 77,455 (for rheumatoid arthritis)

DMF

2.3 Drug Formulation: 5mg, 10 mg, 15 mg, 20 mg, and 25 mg tablets


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3 Studies Submitted Studies Reviewed ADME

Title Study no. Folder/file

1 Pharmacokinetics of INCB018424 in Dogs INCYTE-DMB-06.182.1

M4.2.2.2

2 In vitro and ex vivo protein binding of INCB018424 in rat, cynomolgus monkey, dog, minipig, and human serum and plasma

INCYTE-DMB-07.11.1 M4.2.2.3

3 Quantitatibe whole-body autoradioluminography of rats following oral administration of 14C-INCB018424

INCYTE-DMB-08.58.1 M4.2.2.3

4 Placental transfer and lacteal excretion of 14C-INCB018424 following administration of a single oral dose to Pregnant Sprague Dawley rats

INCYTE-DMB-10.50.1 M4.2.2.3

5 The in vitro metabolism of INCB018424 by rat liver microsomes and individual rat recombinant cytochrome P450(s)

INCYTE-DMB-10.51.2 M4.2.2.4

6 INCB018424: material balance and metabolism in male rats INCYTE-DMB-08.61.1 M4.2.2.4 7 Identification of in vivo metabolites of INCB018424 in female rat following a single

oral dose of 14C-INCB018424 INCYTE-DMB-08.169.1

M4.2.2.4

8 Identification of in vivo metabolites of INCB018424 in beagle dogs INCYTE-DMB-07.14.1 M4.2.2.4 9 Metabolism of [14C]INCB018424 in male and female beagle dogs after a single

oral administration identification INCYTE-DMB-08.149.1

M4.2.2.4

10 Excretion mass balance in female rats and pharmacokinetics of radioactivity in male and female rats following a single oral dose of [14C]INCB018424

INCYTE-DMB-09.82.1 M4.2.2.5

11 Excretion/mass balance in male and female beagle dogs after a single oral administration of [14C]INCB018424

INCYTE-DMB-08.62.2 M4.2.2.5



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Drug interaction Title Study no. Folder/file

name 1 Human CYP Isozymes that are Responsible for the In Vitro Metabolism of

INCB018424 INCYTE-DMB-09.93.1

M5.3.2.2

2 In vitro evaluation o f INCB018424 as an inducer of cytochrome P450 expression in cultured human hepatocytes

INCYTE-DMB-11.06.1

M5.3.2.2

Toxicology studies Repeat dose Title Study no. Folder/file

name 1 A 6-month oral (gavage) toxicity study of INCB018424 in rats with a 6 week recovery period T07-10-06 M4.2.3.2 2 A 6-month oral (gavage) toxicity study with INCB018424 in beagle dogs with 6-week

recovery period T07-10-07 M4.2.3.2

3 52-week oral gavage chronic toxicity and toxicokinetic study with INCB018424 in dogs with a 6-week recovery period

T08-07-03 M4.2.3.2

carcinogenicity Title Study no. Folder/file

name 1 INCB018424: 26 week repeated dose oral carcinogenicity study in Tg.rasH2 mice T09-02-03 M4.2.3.4



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Reproductive toxicology Title Study no. Folder/file

name 1 Oral gavage study of fertility and early embryonic development to implantation with

INCB018424 in rats 8212204

M4.2.3.5

2 Oral administration of INCB018424 via gavage: definitive study for effects on embryo-fetal development in Sprague Dawley rats

T07-12-04 M4.2.3.5

3 Oral administration of INCB018424 via gavage: definitive study for effects on embryo-fetal development in New Zealand white rabbits

T07-12-05 M4.2.3.5

4 Oral gavage study for effects on pre- and post-natal development, Including maternal function with INCB018424 in rats

1001237

M4.2.3.5

Other studies Genetic toxicology Title Study no. Folder/file

name 1 1012571 M4.2.3.7 2 1012575 M4.2.3.7 3 1012576 M4.2.3.7 4 1012577 M4.2.3.7 5 1012578 M4.2.3.7 6 1012579 M4.2.3.7


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Studies submitted, but not reviewed Pharmacology Title Study no. Folder/file

name 1 In vitro activity of INCB018424 metabolites INCYTE-IN

VITRO-07.02.2 M4.2.1.1

2 In vitro activity of INCB018424 in cytokine-mediated cell-based assays INCYTE-IN VITRO-09.08.1

M4.2.1.1

3 In vitro activity of INCB018424 metabolites INCYTE-IN VITRO-069.11.1

M4.2.1.1

4 Pharmacological activity of metabolites of INCB018424 in rats and dogs INCYTE-DMB-07.15.2

M4.2.1.1

5 Pharmacodynamic activity of INCB018424 following a single oral dose in gestating rabbits

INCYTE-IN VITRO-09.13.1

M4.2.1.1

6 Summary report: effect of INCB018424 in rodent models of cancer INCYTE-PRECLIN -10.01.1

M4.2.1.1

7 Summary report: activity of INCB018424 in rodent pharmacodynamic models INCYTE-PRECLIN -10.02.1

M4.2.1.1

8 Summary report: activity of INCB018424 in inflammatory disease models INCYTE-PRECLIN -10.03.1

M4.2.1.1

Secondary Pharmacodynamics Title Study no. Folder/file

name 1 In Vitro Pharmacology: ExpresSProfile- Study of INCB018424" T-06-01-02 M4.2.1.2 2 In Vitro Pharmacology: Kinase Assays- Study of INCB018424 T-06-11-09 M4.2.1.2 3 Binding of INCB018424 to human adenosine A1, A2a and A2b receptors INCYTE-IN

VITRO-07.01.1 M4.2.1.2



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ADME Title Study no. Folder/file

name 1 In vitro permeation of INCB018424 in topical formulations across mouse skin INCYTE-DMB-06.163.1 M4.2.2.2 2 Effect of Elevated Gastric pH on the Oral Absorption of INCB018424

in Cynomolgus Monkeys INCYTE-DMB-09.60.1 M4.2.2.2

3 Pharmacokinetics of INCB018424 in CD-1 Mice After a Single Dose Oral Dose INCYTE-DMB-08.170.1 M4.2.2.2 4 The Pharmacokinetics of INCB018424 in Rats INCYTE-DMB-06.169.1 M4.2.2.2 5 Pharmacokinetics of INCB018424 in Cynomolgus Monkeys INCYTE-DMB-07.08.1 M4.2.2.2 6 Ex vivo and in vitro protein binding of INCB018424 in CD-1 mouse plasma INCYTE-DMB-08.191.1 M4.2.2.3 7 Ex Vivo and In Vitro Protein Binding of INCB018424 in CByB6F1 Hybrid

(Wild-type TgRasH2) Mouse Plasma INCYTE-DMB-08.158.1 M4.2.2.3

8 In Vitro and Ex Vivo Protein Binding of INCB018424 in Rabbit Plasma INCYTE-DMB-09.61.1 M4.2.2.3 9 Brain and cerebrospinal fluid concentrations of INCB018424 in rats INCYTE-DMB-07.12.1 M4.2.2.3 10 Quantitatibe whole-body autoradioluminography tissue distribution of 14C-

INCB018424 in Sprague Dawley rats following a single oral dose INCYTE-DMB-07.109.1 M4.2.2.3

11 Identification of in vivo metabolites of INCB018424 in CD-1 mice after a single oral dose

INCYTE-DMB-08.171.1 M4.2.2.4

12 Metabolism of [14C]INCB018424 in male and female CByB6F1-Tg(HRAS)2Jic mice after a single oral administration

INCYTE-DMB-08.154.1 M4.2.2.4

13 Identification of in vivo metabolites of INCB018424 in female rat following a single oral dose of 14C-INCB018424

INCYTE-DMB-09.84.1 M4.2.2.4

14 Identification of in vivo metabolites of INCB018424 in rabbit INCYTE-DMB-09.95.1 M4.2.2.4 15 Identification of in vivo milk and plasma metabolites of INCB018424 in female

lactating rats following a single oral dose of 14C-INCB018424 INCYTE-DMB-10.51.2 M4.2.2.4



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Drug interaction Title Study no. Folder/file

name 1 Reaction phenotyping: identification of human CYP enzymes involved in the metabolism

of [14C]-VNP401101M NCL-156 M5.3.2.2

2 Metabolic Stability of VNP40101M and VNP4090CE in Human Liver Microsomes and Cryopreserved Human Hepatocytes

NCL-147 M5.3.2.2

3 In vitro metabolism of [14C]VNP40101M in cryopreserved hepatocytes from the rat, dog, and human

NCL-137 M5.3.2.2

Studies reviewed previously and referenced in this review Safety pharmacology Title Study no. Folder/file

name 1 Plethysmography-restrained respiratory assessment of orally administered

INCB018424 to Sprague Dawley rats T06-10-04 M4.2.1.3

2 Toxicokinetics of INCB018424 in rat acute CNS pharmacological profile study T06-10-03 M4.2.1.3 3 Effects of INCB018424 on cloned HERG channels expressed in mammalian cells DMB-06.187.1 M4.2.1.3 4 Cardiovascular assessment of orally administered INCB018424 to conscious

radiotelemetry-instrumented beagle dogs T06-10-01 M4.2.1.3

ADME Title Study no. Folder/file

name 4 The Pharmacokinetics of INCB018424 in Rats INCYTE-

DMB-06.169.1 M4.2.2.2



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Genetic toxicology Title Study no. Folder/file

name 1 INCB018424: Salmonella plate incorporation mutagenicity assay

Key findings: INCB01824 (free base) was not mutagenic in the Salmonella plate incorporation mutagenicity assay, under the conditions of this experiment

AB22ZU.501.BTL M4.2.3.3

2 Salmonella-Escherichia coli/mammalian-microsome reverse mutation assay with a confirmatory assay

T06-08-01 M4.2.3.3

3 INCB018424: Chromosomal aberrations in cultured human peripheral blood lymphocytes

T06-08-02 M4.2.3.3

3 INCB018424: In vivo rat bone marrow micronucleus assay T06-10-02 M4.2.3.3



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4 Pharmacology

4.1 Primary Pharmacology Brief summary

Ruxolitinib is an inhibitor of the Janus Associated kinases (JAKs) with selectivity for JAK1 and JAK2. Ruxolitinib was tested in cell-based assays and in vivo models relevant to the pathogenesis of myeloproliferative neoplasms (MPNs). Activity was observed in in vitro studies and in nonclinical tumor models representing both hematological and solid tumors. Ruxolitinib was shown to inhibit IL-2 stimulated phosphorylation of JAKs and STATs and IL-2 induced proliferation of T cells as well as the phosphorylation of STAT proteins and the production of pro-inflammatory factors (eg, IL-17, IL-22, MCP-1) induced by other cytokines, such as IL-23 and IL-6. Ruxolitinib inhibited splenomegaly in mice resulting from intravenous inoculation of cells expressing the clinically relevant JAK2V617F mutation. The in vitro pharmacological activity of eight ruxolitinib metabolites observed in human plasma was evaluated using enzyme and cell-based assays. The two major human metabolites, M18 and M27 (25% and 11% of parent based on AUC, respectively), as well as the six other metabolites tested were shown to be pharmacologically active. These metabolites had 2-5-fold weaker potency compared to that of the parent compound in a human whole blood assay measuring inhibition of IL-6 induced STAT3 phosphorylation.

In safety pharmacology assessments of ruxolitinib including respiratory and central nervous system studies in rats, and in vitro and in vivo (ECG study in conscious dogs) cardiovascular studies, treatment-related findings were lower body temperature, lower activity, observations of darkened skin and mucous membranes, decreases in minute volume in high dose female rats in the respiratory study and decreases in arterial blood pressure along with increases in heart rate at the highest dose evaluated in dogs in the cardiovascular study.

Primary pharmacodynamics Mechanism of action: Ruxolitinib is a small molecular inhibitor of JAKs with selectivity for JAK1 and JAK2 (IC50 value at 3.3 nM and 2.8 nM respectively). The in vitro studies showed that ruxolitinib inhibited JAK/STAT signaling and growth of a cell line expressing the constitutively active JAK2 mutant (JAK2V617F) that has been implicated in the pathogenesis of the majority of Philadelphia chromosome negative myeloproliferative neoplasms (MPNs). Ruxolitinib treatment resulted in a suppression of phosphorylated STAT3 (pSTAT3) and tumor growth in mice xenograft model with cells expressing JAK2V617F. These results suggest that the alteration of JAK/STAT signaling pathway could contribute to the therapeutic or toxic effect of ruxolitinib.



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Drug activity related to proposed indication: 1- Alteration of JAK/STAT pathway is associated with myeloproliferative disorders The Applicant cited publication Lalentino and Pierre, Biochem Pharmacol (2006) 71: 715 Title: JAK/STAT signal transduction: Regulators and implication in hematological

malignancies This reference article is a commentary review paper; the following summary is copied from above review article. Summary: Signal transducers and activators of transcription (STATs) comprise a family

of several transcription factors that are activated by a variety of cytokines, hormones and growth factors. STATs are activated through tyrosine phosphorylation, mainly by JAK kinases, which lead to their dimerization, nuclear translocation and regulation of target genes expression. Stringent mechanisms of signal attenuation are essential for insuring appropriate, controlled cellular responses. Among them phosphotyrosine phosphatases (SHPs, CD45, PTP1B/TC-PTP), protein inhibitors of activated STATs (PIAS) and suppressors of cytokine signaling (SOCS) inhibit specific and distinct aspects of cytokine signal transduction.

Somatic point mutation in JH2 domain of JAK2 (JAK2V617F), leading also to constitutive tyrosine phosphorylation of JAK2 and its downstream effectors was reported in myeloproliferative disorders.

2- Effects of INCB018424 on kinase activities of JAKs and other enzymes The Applicant cited publication Quintas-Cardama et al. Blood (2010) 115: 3109

Title: Preclinical characterization of the selective JAK1/2 inhibitor INCB018424: therapeutic implications for the treatment of myeloproliferative neoplasms

Summary: INCB018424 inhibited JAK1 and JAK2 with IC50 values of 3.3 nM and 2.8 nM, respectively. The inhibitory effect of INCB018424 on JAK1 and JAK2 was 6 times more effective comparing to Tyk2 and 130 times comparing to JAK3. Methods: The kinase domains of human JAK1 (837-1142), JAK2 (828-1132), JAK3 (781-1124), and Tyk2 (873-1187) were cloned by PCR with N-terminal epitope tags. Recombinant proteins were expressed using Sf21 cells and baculovirus vectors and purified with affinity chromatography. JAK kinase assays used a homogeneous time-resolved fluorescence assay with the peptide substrate (-EQEDEPEGDYFEWLE). Each enzyme reaction was carried out with test compound or control, JAK enzyme, 500nM peptide, adenosine triphosphate (ATP; 1mM), and 2.0% dimethyl sulfoxide



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Summary: Cells expressing JAK2V617F underwent apoptosis when treated with INCB018424. Method: Ba/F3-EpoR-JAK2V617F cells were treated with various concentrations of INCB018424 for approximately 24 hours and analyzed for hallmarks of apoptosis by annexin V/propidium iodide staining or mitochondrial membrane depolarization. Cells treated with INCB018424 were stained with a combination of fluorescein isothiocyanate-annexin V and propidium iodide and analyzed using flow cytometry to determine the percentages of viable, early apoptotic, and late apoptotic or dead cells. The effects of JAK inhibition on mitochondrial membrane potential were determined by flow cytometry using JC-1 as a molecular probe. Results: Treatment with INCB018424 increased apoptosis compared with DMSO, with

a 4.3, 7.2-, and 13.2-fold increase at concentrations of 150, 400, and 1000 nM, respectively.

3-d. INCB018424 inhibited hematopoietic progenitor cell proliferation in primary MPN patient samples

The Applicant cited publication Quintas-Cardama et al. Blood (2010) 115: 3109

Title: Preclinical characterization of the selective JAK1/2 inhibitor INCB018424: therapeutic implications for the treatment of myeloproliferative neoplasms

Summary: INCB018424 inhibited hematopoietic progenitor cell colony formation. Cells derived from patients with polycythemia vera (PV) with mutated JAK2 are more sensitive to JAK1/2 inhibition than normal donors, particularly in the absence of hematopoietic growth factor support. Method: Mononuclear cells from 3 healthy controls and 3 patients with PV expressing the JAK2V617F mutant allele at frequencies more than 90% were obtained. Growth of clonogenic progenitors of erythroid (BFU-E) and myeloid origin (CFU-M) was assessed in colony-forming assays in the presence of increasing concentrations of INCB018424

Results: 1) Inhibition of the growth of erythroid and myeloid progenitors was observed with INCB018424

2) INCB018424 demonstrated potency against erythroid colony formation in JAK2V617F+ human MPNs



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Ruxolitinib was evaluated in a Cerep Kinase Assay screen (T06-11-09) at 0.2 μM

to investigate the effect of ruxolitinib in 30 in vitro kinase assays. Ruxolitinib did not demonstrate significant cross reactivity (defined as > 50% inhibition) against any of the kinases tested in this panel except those of the JAK family: complete inhibition of JAK2 activity was confirmed and JAK3 activity was inhibited by 95%. Note from reviewer: It would have been preferable if a higher concentration of INCB018424 (i.e., 10 μM) had been evaluated in the Cerep ExpresSProfile screen. However, based on the data available for INCB018424, it appears that there is little risk of unintended pharmacological activity due to binding to non-specific receptors/enzymes.

4.3 Safety Pharmacology Safety pharmacology summary:

The safety pharmacology study reports were reviewed by Dr. Barbara Hill for the treatment of rheumatoid arthritis (IND 77,455). Dr. Hill summarized the safety pharmacology study results as follows.

• The NOAEL for central nervous systemic pharmacological effects noted in rats (lower body temperature and reduced total and ambulatory activity counts) following a single oral dose of INCB018424 was identified as 15 mg/kg for males and 50 mg/kg for females, under the conditions of this study;

• The NOAEL for respiratory effects noted in rats (decreased respiratory frequency, increase in tidal volume and increase in minute volume) following a single oral dose of INCB018424 was identified as 15 mg/kg for males and 50 mg/kg for females, under the conditions of this study;

• The IC50 for INCB018424 HERG potassium current was 131.6 μM, under the conditions of this study. The NOAEL for cardiovascular effects noted in male dogs (significant increase in heart rate, significant shortening of PR interval, slight increase in lengthening of the QRS complex, significant shortening of the RR interval and a slight prolongation of the heart-rate corrected QT interval) following a single oral dose of INCB018424 was identified as 10 mg/kg, under the conditions of this study.

Neurological effects: From Dr. Hill’s review

The neuropharmacologic effects of INCB018424 were evaluated in an oral rat GLP study using a Functional Observation Battery (FOB) and locomotor activity measurements. Single oral (gavage) doses of 0, 15, 50 and 150 mg/kg INCB018424 were administered to Sprague-Dawley rats (10/sex/group). Separate toxicokinetic groups for low-, mid- and high-dose groups were included in this study. Toxicology parameters included assessment of mortality. FOB (sensorimotor, neuromuscular and physiological observations) and locomotor activity (measured for a 60 minute interval; home cage, handling and open field observations) were recorded for all animals prior to



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dose administration and 30 minutes post dose (Tmax). Blood samples for toxicokinetic analysis were collected from 6 animals/sex/group at 0, 0.5, 1, 2, 4 and 8 hours post-dose.

No treatment related mortality was noted in this study. No treatment related effects on FOB or locomotor activity were noted in low-dose animals and mid dose females. The only treatment related effect on FOB was noted in high-dose females. A significant lower body temperature was noted in high-dose females (35.1°C) compared to control females (37.1°C) at 30 minutes post-dose. Total and ambulatory activity counts during the first 15 minutes of the 1-hour session were significantly reduced in mid dose males and high-dose animals. A summary of the mean pharmacokinetic parameters for INCB018424 measured in this study is provided in the following table.

A dose-related increase in systemic exposure was noted in this study. Females

exhibited a greater systemic exposure compared to males. The NOAEL for central nervous systemic pharmacological effects noted in rats following a single oral dose of INCB018424 was identified as 15 mg/kg for males and 50 mg/kg for females, under the conditions of this study. Respiratory effects: From Dr. Hill’s review

The respiratory effects of INCB018424 were evaluated in an oral rat GLP study using head-out neck-sealed plethysmography chambers. Single oral (gavage) doses of 0, 15, 50 and 150 mg/kg INCB018424 were administered to Sprague-Dawley rats (8/sex/group). Respiratory parameters included respiratory rate, tidal volume and derived minute volume. Data was captured from 60 minutes pre-dose and continuously for 4 hours post dose.

No treatment-related effects on mortality or clinical signs were noted in this study. A significant decrease in respiratory frequency was noted in high-dose animals. This effect lasted for up to 3 hours post dose with a maximum decrease of 23% in high dose males and 21% in high dose females. A significant increase in tidal volume was noted in mid-dose males and high-dose animals. This effect lasted for up to 4 hours post dose with a maximum increase of 21% in mid-dose males, 34% in high-dose males and 18% in high dose females. The higher tidal volume noted in high-dose animals may reflect a compensatory response to the lower respiratory frequency. A significant decrease in minute volume was noted in high-dose females (up to 17% noted at three timepoints from 0.75 to 1.75 hours post-dose). The NOAEL for respiratory effects noted in rats following a single oral dose of INCB018424 was identified as 15 mg/kg for males and 50 mg/kg for females, under the conditions of this study. Cardiovascular effects:



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From Dr. Hill’s review

The effects of INCB018424 on the cardiovascular system were evaluated in a GLP in vitro HERG assay and in a GLP in vivo cardiovascular safety pharmacology study conducted in Beagle dogs.

INCB018424 (10, 100 and 300 μM) was tested for HERG-channel inhibition in embryonic kidney cells (HEK293). INCB018424 inhibited HERG current by (mean ± SEM): 3.8 ± 0.2% at 10 μM, 40.3 ± 1.6% at 100 μM and by 74.1 ± 0.2% at 300 μM versus 0.6 ± 0.5% for the vehicle control. Significant inhibition of HERG current was noted at 100 and 300 μM INCB018424 compared to vehicle control. The IC50 for INCB018424 HERG potassium current was 131.6 μM, under the conditions of this study. The positive control (60 nM terfenadine) inhibited HERG potassium current by 83.1% in this assay.

Four adult naïve male conscious radiotelemetry-implanted Beagle dogs were used in the in vivo cardiovascular safety pharmacology study. The sponsor states that only male dogs were used in this study because toxicokinetic parameters were similar in male and female dogs. Single oral doses of 0, 3, 10 and 30 mg/kg INCB018424 (dose volume = 10 ml/kg; vehicle: 0.5% methylcellulose) were administered in a Latin-square design. A 3-4 day washout period was incorporated between each dose. Heart rate, arterial blood pressure (systolic, diastolic), body temperature and ECG were collected for a 30-second period every 10 minutes for 24 hours post dose.

No treatment-related effects on mortality were noted in this study. Emesis was

noted after dose administration in high-dose animals. A significantly lower pulse pressure as well as systolic, diastolic and calculated mean pressure (up to 53%, 41%, 31% and 33%, respectively) was noted in high-dose animals compared to control animals. These changes peaked approximately 2-3 hours post dose after which mean arterial blood pressure values began to recover. However, lower values for arterial blood pressure were still noted 24 hours post dose in high-dose animals. No treatment related effects on systolic, diastolic or mean pulse pressure were noted in low and mid-dose animals.

A significant increase in heart rate (increase up to 117% during hour 2) that lasted up to 10 hours post dose was noted in high-dose animals compared to vehicle control. The observed increase in heart rate may reflect a compensatory response to the decreases in arterial blood pressure noted in high-dose animals. No treatment-related effects on heart rate were noted in low- and mid-dose animals. A slight decrease in body temperature was noted at 3 and 4 hours (0.24oC and 0.22oC lower, respectively) in high-dose animals. No treatment-related effects on body temperature were noted in low- and mid-dose animals. A significant shortening of PR interval for up to 6 hours post dose (up to 21% shorter) was noted in high dose animals compared to vehicle control animals. The study report states that these changes in the PR interval were considered to be a consequence of the increases in heart rate. No treatment- related effects on PR interval were noted in low- and mid-dose animals. A slight increase in lengthening of the



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QRS complex for up to 18 hours post-dose (up to 9% or 3.2 msec during hour 3) was noted in high-dose animals compared to vehicle control animals. No treatment related effects on QRS complex were noted in low and mid dose animals. A significant shortening of the RR interval for up to 6 hours post dose (up to 54% shorter during hour 2) was noted in high dose animals compared to vehicle control animals. The study report states that these changes in the RR interval were considered to be a consequence of the increases in heart rate. No treatment related effects on RR interval were noted in low and mid dose animals. A slight prolongation of the heart rate corrected QT interval (QTcV) was noted between hours 11 – 14 (5%) and 19 – 24 (3%) in high- dose animals compared to vehicle control animals. No treatment-related effects on QTcV were noted in low- and mid-dose animals.

Acute cardiovascular effects of INCB018424 after oral administration to high-dose dogs included decreased pulse pressure, systolic, diastolic and mean arterial pressure and increased heart rate. Addition treatment-related effects noted in high-dose dogs included shortening of the RR and PR interval, which may have been related to increased heart rate. A slight lengthening of the QRS complex and corrected QT interval was noted in high dose dogs. The NOAEL for cardiovascular effects noted in male dogs following a single oral dose of INCB018424 was identified as 10 mg/kg, under the conditions of this study. Renal effects: no study conducted Gastrointestinal effects: no study conducted Abuse liability: no study conducted Reviewer comment: The maximum daily dose proposed for BID dosing of ruxolitinib phosphate would be 50 mg/day, equivalent to 29.4 mg/m2/day, based on a 1.7 m2 surface area individual. The NOAEL identified in the conducted safety pharmacology studies (10-50 mg/kg or 60-300 mg/m2 in rats, 10 mg/kg or 200 mg/m2 in dogs) is 2-10 folds of the maximum daily dose proposed. Based on single dose toxicokinetics (rat: INCYTE-DMB-06.174.1; dog: T-07-10-07], the projected Cmax level in female rats at the NOAEL dose of 50 mg/kg is 2.85 μM (0.51 μM unbound), and the projected Cmax level in dogs at the NOAEL of 10 mg/kg is approximately 7.99 μM (0.77 μM unbound), which are 10.4-fold and 15.7-fold, respectively, of the Cmax at the highest proposed therapeutic dose in humans (25 mg bid) associated with a Cmax value of 1.48 μM (0.049 μM unbound, INCYTE-DMB-10.56.3). In addition, the adverse effects observed in the safety pharmacology studies were not noted in the repeated dose toxicology studies. Clinical studies have not recapitulated these findings at clinically relevant doses. Therefore, the potential for ruxolitinib to cause adverse alterations in respiratory, neurologic, and cardiovascular parameters in humans is considered to be low.



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PHARMACOLOGY TABULATED SUMMARY In Vitro Potency of Ruxolitinib in Cell Lines Relevant to MPNs

INCB018424 had anti-tumor effect on the following tested cell lines in vitro- or in vivo

IL-6 dependent INA-6 cell line (plasma cell leukemia cells) TF-1 (leukemia cells) Ba/F3-JAK2V617F cell line (murine leukemia Ba/F3 cells expressing JAK2V617F with the erythropoietin (EPO) receptor) HEL (human erythroleukemia cell line) Progenitor colonies derived from mononuclear cells from patients with PV INA-6 multiple myeloma mouse xenograft model HH human T-cell cutaneous lymphoma xenograft model, mouse Mouse xenograft model with cells expressing Ba/F3-JAK2V617F mutation 22Rv1 Tumor Model, hormone refractory prostate cancer xenograft tumor model, mouse MM1.S Model, myeloma mouse xenograft model

5 Pharmacokinetics/ADME/Toxicokinetics

5.1 PK/ADME Note: PK studies conducted in minipigs are not reviewed. Brief summary

Pharmacokinetic studies were performed in various species after either oral or intravenous administration of ruxolitinib. The PK of ruxolitinib is characterized by a rapid absorption with Tmax value of 0.4-2 h, and a generally short terminal elimination half-life, ranging from 1.5 h to 2 h in rats and dogs. There was no accumulation with 1-month repeated daily dosing, however there was a small accumulation with 6-month



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repeated daily dosing. The absolute oral bioavailability was variable across the nonclinical species, 29% in male rats and 105% in female rats, around 50% in dogs. There were generally no gender differences in exposure of ruxolitinib and its metabolites in dogs. In rats, the Cmax and AUC values were several-fold higher in females compared to males.

In rats, after a single oral dose of 14C-ruxolitinib, drug-derived radioactivity was widely distributed with highest concentration in the GI system, and no detectable levels in the CNS. Ruxolitinib was eliminated from most tissues rapidly and completely. In pregnant rats administered a single oral dose of 14C-ruxolitinib, the fetal:maternal plasma and fetal tissue:maternal plasma concentration ratios were less than one for all fetal tissues indicating that fetal exposure was limited. The plasma protein binding of ruxolitinib was 3.3% in human.

Oxidation was the predominant metabolic pathways in rats and dogs. In vitro studies suggest that CYP3A4 is the predominant CYP isozyme responsible for the metabolism of ruxolitinib. Ruxolitinib is metabolized by isozymes including CPY1A2, CYP2C6 and CYP2D1 found in both male rats and female rats. In rat liver microsomes, ruxolitinib is metabolized to a greater extent in males compared to females, consistent with ruxolitinib being metabolized by the male-rat-specific isozymes CYP2C11, CYP2C13 and CYP3A2.

In rat, dog and human 14C mass balance studies, elimination of drug-derived radioactivity after oral and IV dosing was similarly rapid and complete with excretion via urine, bile and feces. In rats, ruxolitinib-derived radioactivity preferentially partitions into milk with the qualitative metabolite profile being similar to plasma. Milk concentrations were less than 1% of Cmax by 24 h. Ruxolitinib and M18 are not potent inhibitors of CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6 and CYP3A4 in human liver microsomes. In vitro data suggest that ruxolitinib is not an inducer of CYP1A2, CYP2B6 or CYP3A activity at therapeutically relevant concentrations. Methods of Analysis

[See under individual study reviews] Absorption

The pharmacokinetics of INCB018424 in rats

This study (study#INCYTE-DMB-06.169.1) was reviewed by Dr. Barbara Hill. The following summary is excerpted from Dr. Hill’s review.

The pharmacokinetics of INCB018424 was determined in male and female rats given a single 10 mg/kg iv dose (2/sex) or a single 50 mg/kg oral dose (2/sex) of INCB018424 phosphate. The mean systemic plasma clearance of INCB018424 for males (9.4 L/hr/kg) and females (4.8 L/h/kg) were very high and approximated about 3- and 1.5- fold the hepatic blood flow in rats, respectively. The average clearance in males was about 2-fold greater than in females. The mean Cmax and AUC values after oral administration of 50 mg/kg INCB018424 were 2.24 μM and 5.40 μM.hr, respectively, in males and 30.4 μM and 39.3 μM.hr, respectively, in females. This



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equivalent). Blood samples were collected at predose, 15 min, 30 min, 1, 2, 3, 4, 6, 8, 12 and 24 h post-dose in the PO group, and predose, 2.5, 5, 15, 30 min, 1, 2, 3, 4, 6, 8, 12 and 24 h post-dose in the IV group. Analyses for ruxolitinib concentrations in plasma were conducted by liquid chromatographic tandem mass spectrometry (LC-MS/MS). The plasma concentration-time data was used to determine the pharmacokinetic parameters by standard non-compartmental methods using WinNonlin® version 5.0.1 Results: The following figures were copied from the sponsor’s submission Plasma Concentration-Time Profile Following IV (5 mg/kg) or PO (10 mg/kg) Dosing in

Beagle Dogs

PK parameters

Plasma Concentrations (μM) and Pharmacokinetics of INCB018424 in Beagle dogs Given a 10 mg/kg PO Dose or 5 mg/kg IV Dose of INCB018424



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Reviewer comment:

It appears that the sponsor compared the ½ of the AUC from 10 mg/kg oral dosing to the AUC from 5 mg/kg IV dosing to determine bioavailability (57%). Considering the sample size and the PK profile from other conducted studies showing that AUC was not increased in a proportional manner with dose escalation, therefore the bioavailability claimed by the sponsor (57%) may not accurate by using above calculating method.



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Summary of INCB018424Toxicokinetics in Male and Female Beagle Dogs Given Daily Oral Doses of 0.5, 2.5, 5 or 10 mg/kg of INCB018424



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Summary of INCB018424 Toxicokinetics in Male Beagle Dogs Given Daily Oral Doses of 0.75, 1.5, 3 or 6 mg/kg of INCB018424



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Summary of INCB018424 Toxicokinetics in Female Beagle Dogs Given Daily Oral Doses of 0.75, 1.5, 3 or 6 mg/kg of INCB018424



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Methods:

One animal/sex/time point was sacrificed at 2, 8, 24, 168, and 336 hours

postdose. Blood was collected and plasma was harvested at specified time points and carcasses were prepared for quantitative whole-body autoradiography (QWBA). Blood and plasma were analyzed for concentrations of radioactivity using liquid scintillation counting (LSC). Exposed screens were scanned using a Storm scanner for QWBA.

Dosing:

Species/strain: Long Evans, pigmented rats (HsdBlu:LE) #/sex/group or time point: 5/sex Weight: 179-199 g Doses in administered units: 25 mg/kg, 100 μCi Route, form, volume, and infusion rate: oral gavage at dose volume of 10 mL/kg

Results: The following tables are excerpted from Applicant’s submission



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Continued from last page



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In Vitro and Ex Vivo protein binding of INCB018424 in rat, cynomolgus monkey, dog, minipig, and human serum and plasma

Key Study Findings

• In serum: the in vitro unbound fractions of INCB018424 in human, rat, cynomolgus monkey, dog and minipig serum were 3.2, 15, 5.6, 9.5 and 28%, respectively;

• In plasma: the in vitro unbound fractions of INCB018424 in human, rat, dog and minipig plasma were 3.3,1 4, 12 and 26%, respectively;

• The ex vivo unbound fractions of INCB018424 in rat, dog and minipig plasma were 18, 9.7 and 33%, respectively.

Study no: INCYTE-DMB-07.11.1 Volume #, and page #: electronic submission, page 1-11 Conducting laboratory and location: no information provided Date of study initiation: October 20, 2005 GLP compliance: no, QA report: yes ( ) no (x) Drug, lot #, radiolabel, and % purity: INCB018424

Lot No.: 2, 7, 8, 9, 10 and SS-IN2-175; Purity: not provided

Formulation/vehicle: not provided Methods: The in vitro protein binding of INCB018424 was determined using serum

and plasma from rats, cynomolgus monkeys (serum only), dogs, minipigs, and humans. The ex vivo protein binding of INCB018424 was determined using pooled plasma from rats (single oral or IV dose in rat pharmacokinetic study, INCYTE-DMB-06.169.1), minipigs (pharmacokinetic study in Gottingen minipigs administered a single IV dose of 18424, INCYTE-DMB-07.10.1) and dogs (10-day multiple oral dose toxicokinetic study in dogs, INCYTE-DMB-06.185.1). The Multi-Equilibrium Dialyzer SystemTM and diachema membranes from Harvard Apparatus (Holliston, MA) were used for the experiment.

Results: the following tables are excerpted from the Applicant’s submission



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Summary of In Vitro and Ex Vivo Fraction Unbound (fu) of INCB018424 in Serum and Plasma from Human, Rat, Cynomolgus Monkey, Dog and minipig



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days postpartum and analyzed for concentrations of radioactivity using liquid scintillation counting (LSC) to assess the lacteal excretion of 14C-INCB018424-derived radioactivity.

The following summery table is copied from the sponsor.

Dosing:

Species/strain: pregnant Sprague Dawley rats (Hsd:Sprague Dawley SD) Age: 15-17 weeks #/sex/group or time point: see the table in Method section Weight: 234-352 g Doses in administered units: 30 mg/kg, ~50 μCi Route, form, volume, and infusion rate: oral gavage at dose volume of 10 mL/kg

Results: the following tables were excerpted from the sponsor’s submission



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Metabolism The in vitro metabolism of INCB018424 by rat liver microsomes and individual rat

recombinant cytochrome P450(s) Key Study Findings:

• Liver microsomes from male rats metabolized INCB018424 in vitro to a greater extent than females;

• INCB018424 was metabolized by the following isozymes in both female and male rats; CYP1A2, CYP2C6 and CYP2D1

• INCB018424 was metabolized extensively by the male specific rat CYP isozymes CYP2C11, CYP2C13 and CYP3A2.

Study number: INCYTE-DMB-07.01.2-153 Volume #, and page #: Electronic submission; page 108 M4/42-stud-rep/422-pk/4224-metab/ncl-153.pdf, page 1-43 Conducting laboratory and location: Incyte Corporation Date of study initiation: August 7, 2006 GLP compliance: no, QA report: yes ( x ) no (x) Drug, lot #, radiolabel, and % purity: INCB018424 Lot SS-IN2-175 Purity: not provided Formulation/vehicle: 50 mM potassium phosphate buffer (pH 7.4), 2mM NADPH, Methods: Metabolism by Rat Liver Microsomes

INCB018424 was incubated in triplicate with either female or male rat liver microsomes at 37°C. Each incubation contained 0.5 mg/mL of the either female (pool of 10 rats) or male (pool of 10 rats) rat liver microsome, 50 mM potassium phosphate buffer (pH 7.4), 2mM NADPH, and INCB018424 (1 μM). Aliquots were taken at 0, 10, 20 and 30 minutes, deproteinized with acetonitrile and stored at -20°C until analysis.

Metabolism by Individual Rat Recombinant CYP Isozymes INCB018424 was incubated in triplicate with individual rat cytochrome P-450 (CYP) isozymes at 37°C. Each incubation contained 5 pmols of the individual isozyme, 50 mM potassium phosphate buffer (pH 7.4), 2mM NADPH, and INCB018424 (1 μM). Aliquots were taken at 0, 30 and 60 minutes, deproteinized with acetonitrile and stored at -20οC until analysis.

Results: The Applicant only presented the summary table. The following summary tables are excerpted from the Applicant’s submission.



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Date of study initiation: March 20, 2007 GLP compliance: no, QA report: yes ( ) no (x) Drug, lot #, radiolabel, and % purity: INCB018424 Lot SS-IN2-175 Purity: not provided [14C]INCB018424 Lot 3562192 Purity: 99% (radiochemical purity) Formulation/vehicle: 50 mM potassium phosphate buffer (pH 7.4), 2mM NADPH, Methods: INCB018424 was administered by the oral and intravenous routes to male Crl:CD(SD) rats. Dose solutions were prepared as follows:

Study design is summarized in the following table:

Following dosing, rats were housed individually in metabolism units suitable for

the collection of urine and feces up to a maximum of 4 days. Blood samples were collected pre-dose, and at 5, 15, 30, 60, 90, and 120 minutes post-dose for the intravenous group, and 0.5, 1, 2, 4, 5, 6, 7, and 8 hours post-dose for the oral group. Urine, feces, bile and/or plasma samples were analyzed by HPLC and mass spectroscopy to determine the molecular weight of any radioactively labeled metabolites.

Results: the following figures and tables are excerpted from the Applicant’s

submission.



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Identification of rat plasma metabolites of INCB018424 after a single oral dose of 14C-INCB018424

Key study findings:

• INCB018424 was present in plasma from the 1 and 4 hour timepoints, with 12 radiochromatographic peaks related to INCB018424 observed;

• A total of seven metabolites of INCB018424 were identified and confirmed using previously isolated or synthesized metabolites of INCB018424;

• The two most abundant circulating plasma metabolites were INCB025258 (M8), a 3-hydroxyl metabolite of the cyclopentyl ring and INCB025256 (M11), a 3-oxocyclopentyl metabolite.

Study no: INCYTE-DMB-08.169.1 Volume #, and page #: electronic submission, page 1-43 Conducting laboratory and location: INCYTE Date of study initiation: September 13, 2008 GLP compliance: no, QA report: yes ( ) no (x) Drug, lot #, radiolabel, and % purity: INCB018424 Lot SS-IN2-175 Purity: not provided

14C-INCB018424 Lot 3562192 Purity: not provided Formulation/vehicle: not provided Methods: Plasma samples from male rats orally dosed with 25 mg/200 μCi/kg 14C-

INCB018424 were obtained on day 1 of a single dose tissue distribution study conducted at study # 59N-070. Residual plasma samples collected 1, 4 and 24 h post-dose from each rat were pooled by time point. Samples were assayed using electrospray ionization LC-MS with a Thermo Finnigan LCQ Deca-XP Plus Ion-Trap Mass Spectrometer (Thermo-Fisher Scientific Waltham MA), operated in positive ionization mode.


(b) (4)


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Source of Study Samples for Biotransformation Experiments

Results:

Summary Table of Metabolites of INCB018424 Found in Male Rat Plasma at 1 Hour Post-Dose



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Summary Table of Metabolites of INCB018424 Found in Male Rat

Plasma at 4 Hours Post-Dose

Identification of in vivo metabolites of INCB018424 in female rat following a single oral dose of 14C-INCB018424

Key study findings:

• INCB018424 was present in plasma from female rat at the 1, 2, and 4 hour timepoints, with a total of 18 radiochromatographic peaks related to INCB018424 observed;

• Concentrations of metabolites in plasma were low (less than 0.8 μg) equivalents/g in all cases;

• There were eight circulating plasma metabolites present at 1 or 2 hours post-dose at levels that were >10% of parent (based on radiochemical detection) in order of decreasing amount: INCB025258 (M8); INCB025256 (M11); INCB018424 (M7),

• Structures for INCB025257 (M7); INCB032568 (M48); INCB025264(M16); INCB027598 (M18); INCB025262 (M27); and M35 have not been elucidated;

• The overall metabolic profile is similar in males and females, with the three most abundant metabolites of INCB025258 (M8), INCB025256 (M11), and INCB025257 (M7), in decreasing amount, respectively.

Study no: INCYTE-DMB-08.169.1



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Volume #, and page #: electronic submission, page 1-120 Conducting laboratory and location: INCYTE Date of study initiation: September 22, 2009 GLP compliance: no, QA report: yes ( ) no (x) Drug, lot #, radiolabel, and % purity: INCB018424 Lot BPR-07-101-B2-21 Purity: not provided 14C-INCB018424 Lot 65359001 Purity: not provided Formulation/vehicle: not provided Methods:

Source of Study Samples for Biotransformation Experiments

Plasma, bile, urine, feces obtained from either male or female Sprague-Dawley

rats were prepared by pooling each sample obtained from individual animals at the following collection time points: Plasma: 1, 2, 4, 8, 24, hours for females; 1, and 4 hours for male rats Urine, bile and feces: 0-8, 8-24, 24-48, 48-72, 72-96 hours for female rats

Samples were assayed using electrospray ionization LC-MS with a Thermo Finnigan LCQ Deca-XP Plus Ion-Trap Mass Spectrometer (Thermo-Fisher Scientific Waltham MA), operated in positive ionization mode. Results:



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Quantitative Summary (as % of Parent Present and μg equivalent/g) of Metabolites of 14C-INCB018424 Found in Female Rat Plasma Following a Single 25 mg/kg Oral Dose



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Quantitative Summary (as % of Administered Radioactivity) of Metabolites of 14C-INCB018424 Found in Female Rat Excreta Following a Single 25 mg/kg Oral Dose



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Quantitative Summary of Metabolites of 14C-INCB018424 (as μg equivalents/g and % of Administered Dose) Found in Female Rat Urine Following a Single 25 mg/kg Oral Dose

(0-24 Hour)



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Quantitative Summary of Metabolites of 14C-INCB018424 (as μg equivalents/g and % of Administered Dose) Found in Female Rat Bile Following a Single 25 mg/kg Oral Dose

(0-24 Hour)



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Quantitative Summary of Metabolites of 14C-INCB018424 (as μg equivalents/g and % of Administered Dose) Found in Female Rat Feces Following a Single 25 mg/kg Oral Dose

(0-24 Hour)



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Identification of in vivo metabolites of INCB018424 in beagle dogs

Key study findings:

• INCB018424 underwent extensive metabolism, and the major metabolites were derived from oxidation and subsequent glucuronidation.

Study no: INCYTE-DMB-07.14.1 Volume #, and page #: electronic submission, page 1-48 Conducting laboratory and location: INCYTE Date of study initiation: January 7, 2007 GLP compliance: no, QA report: yes ( ) no (x) Drug, lot #, radiolabel, and % purity: INCB0018424 Lot # SS-IN2-175 Formulation/vehicle: not provided Methods: The plasma and urine samples were obtained from the beagle dogs following a single intravenous administration of INCB018424 at 5 mg/kg (Report: INCYTE-DMB-06.182.1). The residual plasma samples from this pharmacokinetic study were pooled from individual dogs and time points to generate samples of ample volume to characterize the metabolic profile. Samples were analyzed by liquid chromatography tandem mass spectrometry (LC/MS/MS).



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Drug, lot #, radiolabel, and % purity: INCB018424 Phosphate Lot # SS-IN2-175 Purity: 99% [14C]INCB018424 Lot#356219 Purity: 99% Formulation/vehicle: not provided Methods:

Selected plasma, urine and fecal samples were analyzed by HPLC for metabolite

characterization.

Excretion

Excretion Mass Balance in Female Rats and Pharmacokinetics of Radioactivity in Male and Female Rats Following a Single Oral Dose

of [14C]INCB018424 Key Study Findings:

• Elimination of INCB018424-derived radioactivity occurred via the urine, bile and feces, accounting for an average of 45%, 40% and 20% of the administered dose, respectively;

• Excretion of radioactivity in urine and bile was rapid, with 40% of the dose recovered in the 0-8 h urine specimen, 37% in the 0-8 h bile specimen and 19% in the 0-24 h feces specimen

• The pharmacokinetics of blood and plasma total radioactivity were similar;



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Bile, urine, feces, and plasma samples were collected at the time points shown in the table

below: Sample Collection Summary

The radioactivity content was then quantified by LSC. All LSC analyses were

performed using a Model 2800TR liquid scintillation analyzer (Perkin Elmer), counting for at least 5 min or 100,000 counts. Dosing:

Species/strain: Sprague-Dawley rats #/sex/group or time point: see the table above Age: not provided Weight: 242-256 g Doses in administered units: 25 mg/kg; 200 μCi/rat Route, volume: oral gavage at dose volume of 10 mL/kg

Results: The following figure was submitted by the Applicant



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Recovery of Radioactivity for Group 1 Female Rats Following Oral Administration of [14C]INCB018424 at a Target Dose of 25 mg/kg



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Plasma and Blood Concentrations of Total Radioactivity in Group 2 Female Rats Following Oral Administration of [14C]INCB018424 at a Target Dose of 25mg/kg



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Plasma and Blood Concentrations (Mean and SD, μg equivalents/mL) of Total Radioactivity in Group 2 Female and Group 3 Male Rats Following Oral

Administration of [14C]INCB018424 at a Target Dose of 25 mg/kg



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Time Course of Excretion of Radioactivity by Group 1 (Bile Cannulated) Female Rats Following Oral Administration of [14C]INCB018424 at a Target Dose of 25 mg/kg



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Dosing:

Species/strain: Beagle dog #/sex/group or time point: see the table above Age: 6 to 12 months Weight: 7.9 to 9.8 kg Doses in administered units: 3 mg/kg, 10 μCi/kg Route, volume: oral gavage at dose volume of 10 mL/kg

Results (presented for total radioactivity only):

[14C]INCB018424 Radioactivity in Blood Cells Collected from Male and Female Beagle Dogs after a Single PO Administration of INCB018424 at Approximately 3

mg/kg Body Weight



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[14C]INCB018424 Radioactivity in Plasma Collected from Male and Female Beagle Dogs after a Single PO Administration of INCB018424 at Approximately 3 mg/kg

Body Weight

Total Recovery of Dosed Radioactivity in Male and Female Beagle Dogs after

Administration of INCB018424 at Approximately 3 mg/kg Body Weight


BEST AVAILABLE

COPY


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Methods: Three preparations of cultured human hepatocytes (two cryopreserved and one fresh) from three separate livers were treated once daily for three consecutive days with dimethyl sulfoxide (DMSO, 0.1% v/v, vehicle control), one of three concentrations of INCB018424 (1, 3 or 10 μM) or one of two known human CYP inducers, namely, omeprazole (100 μM) and phenobarbital (750 μM). After treatment, the cells were treated in situ with the appropriate marker substrates for the analysis of phenacetin O-dealkylation (marker for CYP1A2) and bupropionhydroxylation (marker for CYP2B6) by LC/MS/MS. Discussion and Conclusions

The absorption, distribution, metabolism and excretion of ruxolitinib has been studied in mice (CD-1, hairless and transgenic), rats (albino and pigmented), rabbits, dogs, monkeys and minipigs. Summary and conclusion below is mostly based on the study results from rat and dog studies.

Oral pharmacokinetics studies have shown that ruxolitinib was absorbed rapidly with Tmax values ranging from 0.45 to 2 h; the terminal elimination half-life of ruxolitinib was generally short after oral and IV dosing, ranging from 1.5 h (rat) to 2 h (dog). The absolute oral bioavailability was variable across the nonclinical species, 29% in male rats and 105% in female rats, and approximately 50% in dogs. No accumulation was observed after 1-month repeated daily dosing, while small accumulation was observed after 6-month daily administration of ruxolitinib (the plasma exposures at the end of 6-month treatment were about 2-fold of those observed on study day 1). There were generally no gender differences in the exposure of ruxolitinib and its metabolites in dogs. In rats, the Cmax and AUC values were several-fold higher in females compared to males, in part due to higher metabolic clearance in male rats. In addition, the human metabolites of ruxolitinib were identified and observed at similar or higher exposures compared to human during the course of nonclinical toxicology studies in mice, rat and dog and in the carcinogenicity study in Tg.rasH2 mouse. The terminal half-life, routes and rate of excretion for ruxolitinib-derived radioactivity and metabolites observed between male and female rats were similar. Following IV administration of ruxolitinib, the total systemic clearance ranged from moderate in dogs to very high in rats. Following IV administration of ruxolitinib, the total systemic clearance and volume of distribution was species-dependent and ranged from moderate in dogs (26% of hepatic blood flow in dogs) to very high in rats (greater than 100% of hepatic blood flow). The apparent steady-state volume of distribution (Vss) of ruxolitinib in male rats, female rats, and dogs was 3.8, 1.6, and 1.1 L/kg, respectively, indicating ruxolitinib is not distributed extensively beyond body water in dog (consistent with humans), and may be distributed to a greater extent in rat. Ruxolitinib is not distributed significantly beyond body water in dog and monkey and may be distributed to a greater extent in rats.

In rats, after a single oral dose of 14C-ruxolitinib, drug-derived radioactivity was widely distributed with rapid and complete elimination from most tissues. High concentration of ruxolitinib was detected in the GI system, levels in the CNS were below LOQ. Ruxolitinib was eliminated from most tissues rapidly and completely. In pregnant rats administered a single oral dose of 14C-ruxolitinib, fetal exposure was observed but



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was less than maternal exposure with fetus:maternal plasma and fetal tissue:maternal plasma concentration ratios being less than one for all fetal tissues. The plasma protein binding of ruxolitinib is species-dependent 3.3% in human. In rat and dog mass balance studies, elimination of drug-derived radioactivity after oral and IV dosing is similarly rapid and complete with excretion via urine, bile and feces. In rats, ruxolitinib-derived radioactivity preferentially partitions into milk with the qualitative metabolite profile being similar to plasma. Milk concentrations were less than 1% of Cmax by 24 h. In milk, the qualitative metabolite profile was similar to plasma, with quantitative differences likely due to the higher exposure of ruxolitinib-related radioactivity in milk relative to plasma.

Oxidation was the predominant metabolic pathways in rats and dogs. In vitro

studies suggest that CYP3A4 is the predominant CYP isozyme responsible for the metabolism of ruxolitinib. Ruxolitinib is metabolized by isozymes including CPY1A2, CYP2C6 and CYP2D1 found in both male rats and female rats. In rat liver microsomes, ruxolitinib is metabolized to a greater extent in males compared to females, consistent with ruxolitinib being metabolized by the male-rat-specific isozymes CYP2C11, CYP2C13 and CYP3A2. For the major human metabolites (M18 and M27) and most of the measured minor metabolites, they were studied in one or more animal species at the exposure levels exceeding or approaching corresponding human exposure at the highest proposed therapeutic dose (25 mg twice daily).

Tables and figures to include comparative TK summary The following tables are from the Applicant. Some of the studies summarized in the table (e.g. mouse, minipig, monkey studies) are not reviewed.



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6 General Toxicology Overall toxicology summary General toxicology: Single- and repeat-dose toxicology studies up to 6 months in duration in rats and up to 12 months in duration in dogs were conducted. Single doses of ruxolitinib up to 100 mg/kg in rats and 40 mg/kg in dogs were well tolerated; these were the highest doses tested in these studies. No ruxolitinib-related acute toxicity was determined in the single dose studies. Of note single-dose studies were non-GLP, only limited dose levels were used and minimal toxicity evaluations were performed. Repeated dosing in rats and dogs resulted in toxicities mainly in the hematopoietic system including reduced levels of circulating leukocytes, especially lymphocytes in rats and dogs. Lymphoid depletion was noted in multiple lymph nodes and spleen. Genetic toxicology: Ruxolitinib was not mutagenic in the bacterial mutagenicity assay or clastogenic in the in vitro chromosome aberration assay or in the in vivo micronucleus assay in rats. Carcinogenicity: Ruxolitinib was not carcinogenic in a 26-week study in Tg.rasH2 mice. A 2-year rat carcinogenicity study is ongoing. Reproductive toxicology: In embryo-fetal developmental studies in the rat and rabbit, ruxolitinib was not teratogenic, but was associated with increased late resorptions (rabbit only) and fetal toxicity (reduced fetal weights in rats and rabbits). The No-Observed-Adverse-Effect-Level (NOAEL) dose for the rat and rabbit study was 30 mg/kg/day. In a pre- and post-natal development study, there were no adverse findings for fertility indices and maternal and embryo-fetal survival, growth, and developmental parameters. A placental transfer and lactation study in rats given ruxolitinib demonstrated that low concentrations of ruxolitinib cross the placenta, and parent compound is eliminated rapidly resulting in limited fetal exposure. In lactating rats, ruxolitinib derived radioactivity was excreted into the milk with a concentration that was 13-fold higher than the corresponding maternal plasma concentration. Special toxicology: no study conducted

6.1 Single-Dose Toxicity Some single-dose studies were reviewed by Dr. Babara Hill under IND 77101, the results are not presented here. In conclusion, the toxicities associated with ruxolitinib were similar to those observed in the repeated-dose studies.



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Methods Doses: 5,10,30, 60 mg/kg*

* The dose levels were stated to be based on previous studies; no other specific information was provided

Frequency of dosing:

once daily for 182 days, followed by a 42-day recovery period (recovery group)

Route of administration:

Oral gavage

Dose volume: 10 mg/mL Formulation/V

ehicle: 0.5% methylcellulose in reverse osmosis-treated water

Species/Strain:

Rat/Crl:CD(SD)

Number/Sex/Group:

See table below

Age: approximately 35 days old Weight: ranged from 199 g to 286 g for males and from 139 g to 199 g for femalesSatellite groups:

5/sex for control, 11/sex/group for treatment group

Unique study design:

none

Deviation from study protocol:

none

Number of Animals/sex/group- Rising Dose

Vehicle 0 mg/kg/day 5 mg/kg/day 10 mg/kg/day 30 mg/kg/day 60 mg/kg/day

Main 15 15 15 15 15 Recovery 8 8 8 8 8 Observations and Results OBSERVATIONS AND TIMES:

Mortality Two times daily Clinical examinations Two times daily Detailed physical examinations weekly Body weights weekly Food consumption weekly Ophthalmoscopy Weeks -1, 25, 32 Clinical Pathology: week 12 (hematology and serum

chemistry), and prior to sacrifice (week 26 or 32)

Gross pathology: At death or at scheduled sacrifice Organ weights: At sacrifice Histopathology: At sacrifice or death

Adequate Battery: yes (x), no ( ) , Peer review: yes (x), no ( )



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Toxicokinetics: prior to dosing and at 0.5, 1, 2, 4, 6, 8, 12 and 24 hours after dose administration on study days 0, 27 and 181.

RESULTS: Mortality Animal Sex Dose

(mg/kg/day) Days of death

Type of death

Death cause

8177 M 0 57 Found dead unknown 8078 M 5 162 Found dead unknown 8198 M 30 155 Found dead unknown 8222 M 30 166 Found dead unknown

8331 F 60 101 Found dead unknown

Summary: None of the early death are considered as treatment-related deaths. Clinical Signs

Early death animals • unremarkable 5 mg/kg/day • unremarkable 10 mg/kg/day • unremarkable 30 mg/kg/day • unremarkable 60 mg/kg/day • wet clear and wet/dry red material around

the mouth, male and female Body Weights The following figures are excerpted from the Applicant’s submission, the data are verified by the reviewer.



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Summary of body weight (g) (dosing period- males)



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Summary of body weight (g) (dosing period- Females)

Summary of body weight (g) (dosing period- females)



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Summary: • During the dosing period, decreased body weights were observed in test article-

treated group males as early as the second week of dosing. Mean body weights at the end of the dosing period were 1.8%, 4.4%, 6.0% and 11.7% lower than control values for the 5, 15, 30 and 60 mg/kg/day group males, respectively. Lower weight changes were partially recovered by the end of the recovery period.

• No significant body weight changes were observed in females. Feed Consumption Lower food consumption (occasionally statistically significant) was noted for the 60

mg/kg/day group males (↓0- 21%). A similar trend was not observed for the 60 mg/kg/day group females.

Ophthalmoscopy No test-article related changes Hematology

MALES Percent Change From Vehicle Control Doses (mg/kg/day) 5 15 30 60 Assessment week 12 26 32 12 26 32 12 26 32 12 26 32

Hemoglobin ↓7 ↓4 hematocrit ↓7 ↓3 WBC ↓12 ↓5 ↓18 ↓30 ↓16 ↓29 ↓24 ↓18 ↓32 ↓32 ↓14 Neutrophil (E3/μl) ↓9 ↓6 ↓23 ↓8 ↑39 ↑15 ↓12 ↑45 ↑12 ↓6 Lymphocyte (E3/μl) ↓13 ↓5 ↓16 ↓33 ↓20 ↓8 ↓36 ↓31 ↓19 ↓45 ↓43 ↓16 Monocyte (E3/μl) ↑10 ↓20 ↑18 ↑10 ↓5 ↓5 ↓23 ↑10 ↓14 ↓13 Eosinophil (E3/μl) ↑8 ↓21 ↓25 ↓8 ↓7 ↓42 ↑23 ↑25 ↓8 ↑7 Basophil (E3/μl) ↓33 ↓50 ↓33 ↓33 ↓57 ↓14 ↓63 ↓57 ↓14 LUC (E3/μl) ↓12 ↓28 ↓38 ↓14 ↓63 ↓44 ↓25 ↓57 ↓13

FEMALES Percent Change From Vehicle Control Doses (mg/kg/day) 5 15 30 60 Assessment week 12 26 32 12 26 32 12 26 32 12 26 32

Hemoglobin ↓4 ↓2 WBC ↓6 ↓17 ↑8 ↓21 ↓10 ↓19 ↓21 ↓39 ↓38 Neutrophil (E3/μl) ↑21 ↓6 ↓8 ↑30 ↓6 ↑22 ↓8 ↓8 ↑44 ↓8 Lymphocyte (E3/μl) ↓9 ↓19 ↑12 ↓24 ↓18 ↓25 ↓23 ↓50 ↓46 ↑10 Monocyte (E3/μl) ↓25 ↓14 ↓10 ↑7 ↓30 ↓15 ↓7 ↓40 ↓20 Eosinophil (E3/μl) ↑33 ↓11 ↑11 ↓22 ↓43 ↓44 Basophil (E3/μl) ↓50 ↓50 ↓50 ↓100 ↓50 ↓100LUC (E3/μl) ↓20 ↓40 ↓40 ↓40 ↑33 ↓40 ↓60 ↑33 ↓60 ↓60

Blank cells: unremarkable changes. Week 32 represents the recovery data. LUC: large unstained cell.



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Summary: • Lower white blood cell counts and lower counts of absolute, lymphocyte,

monocyte, basophil, and large unstained cells (LUC) were noted at study weeks 12 and 26 in male and female animals at all dose levels of INCB018424. Hemoglobin and hematocrit were slightly lower in the male and female groups at 60 mg/kg/day. These changes were reversible.

Clinical Chemistry No test-article related changes Urinalysis No test-article related changes Gross Pathology Unremarkable Organ Weights The following table is excerpted from the Applicant’s application. The data on the table are verified by the reviewer.

Toxicologically Relevant Final Body Weight and Organ Weight Changes At Study Week 26



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Toxicologically Relevant Final Body Weight and Organ Weight Changes At Study Week 32 (recovery)

Dose Group (mg/kg/day): 5 15 30 60 Final Body Weight (male) ↓9% ↓11% ↓18% ↓13% Final Body Weight (female) ↓5% ↓5% Adrenal Gland (Male) Absolute ↑4% ↓13% ↓2% ↓8% Relative-to-Body ↑25% ↑25% ↑13% Relative-to-Brain ↓13% ↓9% Spleen (Male) Absolute ↑6% ↑12% Relative-to-Body ↑17% ↑25%** ↑20% ↑17% Relative-to-Brain ↑13% Spleen (Female) Absolute ↓7% ↓5% ↓7% Relative-to-Body ↓5% Relative-to-Brain ↓6% Note: Blank: unremarkable change Summary:

• Test article-related changes include decreased weights of the spleen in both males and females, and decreased weights of adrenal gland in males. Organ weight changes were reversible.

Histopathology At the study week 26, test article-related histologic changes were restricted to the 60 mg/kg/day group where minimal to mild lymphoid depletion was observed in the spleen and mandibular lymph nodes of both genders. Additionally, minimal cortical atrophy was observed in the adrenal glands of the 60 mg/kg/day group males. The incidence and severity of microscopic findings in the spleen, mandibular lymph nodes, and adrenal glands are summarized in the following table (from the submission). At the study week 32 (recovery necropsy), microscopic changes were not observed that were attributed to the test article, suggesting full recovery.



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Incidence Of Selected Histopathologic Findings At The

Study Week 26 Primary Necropsy

Toxicokinetics: refer to PK section. STUDY SUMMARY:

INCB018424 administered by oral gavage to Sprague Dawley rats at doses of 5, 15, 30 and 60 mg/kg/day for 26 weeks was well tolerated, with no test article-related deaths, ophthalmic findings or alterations in coagulation and urinalysis parameters. Test article-related clinical observations were limited to findings of clear and red material around the mouth noted for the 60 mg/kg/day group males and females. Lower body weights were noted in a dose-related manner for treated males and lower food consumption was noted for the 60 mg/kg/day group males. The mean body weight for the 60 mg/kg/day males was approximately 12% lower than the mean control value by the end of the dosing period and did not fully recover by the end of the 42-day recovery period. Reduced levels of circulating WBCs and lymphocytes were noted at all dose levels along with lower spleen weights in both sexes. Lymphoid depletion was documented in most spleen sections and in several mandibular lymph nodes at the 60 mg/kg/day dose level. Adrenal atrophy observed in histopathology correlated with reduced absolute and relative weight of adrenal glands. Clinical and anatomical pathology findings at the study week 32 recovery necropsy indicated that partial to full recovery was in progress in both genders administered all dose levels of INCB018424.



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Number/sex/group: Number of Animals/sex/group Vehicle

Control 0.5 mg/kg/d

2.5 mg/kg/d

5.0 mg/kg/d

10mg/kg/d

Main 5 5 3 3 5 Recovery 1-2 1-2 1-2 1 -2 1-2

Route, formulation, volume Oral gavage. diluted in 0.5% methylcellulose in reverse osmosis-treated water, 10 mg/mL

Satellite groups used for toxicokinetics:

12/sex/group

Study design: Male and female dogs were dosed daily via oral gavage for a total 6 months, followed by a 43 days recovery period (recovery groups)

OBSERVATIONS AND TIMES:

Mortality Two times daily Clinical examinations daily Detailed physical examinations weekly Body weights weekly Food consumption Recorded daily, reported weekly Hematology and coagulation prior to randomization (study day -5) and on

study days 27, 49, 86, 135 (hematology only), 174 and 224

Serum chemistry prior to randomization (study day -5) and on study days 27, 49, 86, 174 and 224

Urinalysis prior to randomization (study day -5) and on study days 27, 49, 86, 174 and 224

Urine metabolite profiling on study days -8, 0 and 176 Ophthalmoscopy during study weeks -2, 25 and 32 EKG weeks -2, 25 and 31 Gross pathology: All animals sacrifice Organ weights: All animals at sacrifice Histopathology: animals found dead or euthanized in

extremis as well as all animals in the control (0 mg/kg/day) and high dose (10 mg/kg/day) groups. Adequate Battery: yes (x), no ( ) , Peer review: yes (x), no ( )

Toxicokinetics: prior to dosing and at 0.5, 1, 2, 4, 8, 12 and 24 hours after dose administration on study days 0, 40 and 175



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RESULTS: Mortality: Three early deaths at 10 mg/kg/day. 1 female (# 4454): died on study day 126, 2 males: # 4411 was found dead on study day 179, # 4430 was euthanized in extremis on study day 156. Clinical signs: Early death: Male #4411: reddened facial area, clear to colored discharge from the eyes, soft

feces and/or diarrhea were observed prior to death. Male #4430: hypoactivity, prostration with labored respiration and swelling in the left

hindlimb were noted. Other clinical observations noted prior to euthanasia included soft feces, diarrhea and clear and/or colored discharge from the eyes and warts on the left hindlimb.

Female #4454: Clinical observations prior to death included hypoactivity, increased respiration rate, labored respiration, diarrhea, swelling of the left and right forelimbs and hindlimbs and impaired use of the left and right hindlimbs.

Terminal death: Test article-related clinical observations consisting of clear to colored discharge

from the eyes, soft feces and/or diarrhea (females), reddened areas, swelling and hair loss on the limbs (which correlated to demodectic mange), were noted for the 5 and 10 mg/kg/day group males and females

Several animals in the 10 mg/kg/day group were also observed with interdigital cysts. Additionally, four animals at this dose had warts on the limbs and/or facial area.

Bodyweight: With the exception of the early death animals in the 10 mg/kg/day group

which had some notable body weight changes (male #4430 lost 0.7 kg total; female #4454 lost 0.7 kg total), there were no obvious test article-related changes in mean body weights in any treated group when compared to the control group.

Food Consumption: < week 19: unremarkable 19-26 weeks: male: lower at 10 mg/kg, ↓8%-28% comparing to control group female: dose-dependently reduced in all treatment groups 0.5 mg/kg-↓11%-22% comparing to control group 2.5 mg/kg- ↓4%-18% comparing to control group 5 mg/kg- ↓11%-17% comparing to control group 10 mg/kg--↓11%-28% comparing to control group 26-32 weeks: male: lower at 10 mg/kg, ↓31%-51% comparing to control group female: 0.5 mg/kg-↓10%-42% comparing to control group 2.5 mg/kg- ↓41%-↑9% comparing to control group 5 mg/kg- ↓12%-37% comparing to control group 10 mg/kg--↓42%-↑17% comparing to control group



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Hematology:

MALES Percent Change From Vehicle Control

5 mg/kg/day Assessment Day 27 49 86 135 174 224 RBC ↓8 ↓4 ↓10 ↓9 ↓14 Hemoglobin ↓6 ↓10 ↓9 ↓16 Hematocrit ↓7 ↓10 ↓9 ↓15 Reticulocyte ↓27 ↓29 ↓25 ↓29 ↑15 ↓10 Neutrophils ↑29 ↑23 ↓6 ↑5 ↑9 ↓5 Lymphocytes ↓13 ↓11 ↓11 ↓14 Monocytes ↑49 ↑32 ↓17 ↑15 ↑31 ↑43 Eosinophils ↓17 ↓18 ↓59 ↓35 ↓36 Basophils ↓50 ↓50 ↑50

10 mg/kg RBC ↓11 ↓13 ↓16 ↓20 ↓27 ↓9 Hemoglobin ↓10 ↓13 ↓18 ↓22 ↓33 ↓17 Hematocrit ↓11 ↓14 ↓17 ↓21 ↓30 ↓16 Reticulocyte ↓32 ↓17 ↓30 ↓12 ↑22 ↑11 Neutrophils ↑22 ↑16 ↑30 ↑71 ↑64 ↓23 Lymphocytes ↓21 ↓29 ↓23 ↓13 ↑28 ↓33 Monocytes ↑37 ↑21 ↑46 ↑70 ↑102 ↑60 Eosinophils ↓48 ↓53 ↓64 ↓30 ↓59 ↓29 Basophils ↓25 ↓33 ↓50 ↑8 ↑38 LUC ↑20 ↑100 ↑50 ↓63

FEMALES Percent Change From Vehicle Control

5 mg/kg/day Assessment Day 27 49 86 135 174 224 RBC ↓8 Hemoglobin ↓6 ↓9 Hematocrit ↓5 ↓9 Reticulocyte ↓18 ↓18 ↓30 ↑47 ↓21 Monocytes ↓10 ↑12 ↑20 ↓26 Eosinophils ↓41 ↓68 ↓57 ↓49 ↓80 ↑74 Basophils ↑67 ↑67 ↑100 ↑50 ↑100 LUC ↑85 ↑71 ↑63 ↑113 ↑83

10 mg/kg/day RBC ↓6 ↓7 ↓14 ↑8 Hemoglobin ↓5 ↓ 8 ↓11 ↓11 ↓19 Hematocrit ↓6 ↓9 ↓10 ↓17 Reticulocyte ↑5 ↓15 ↓12 ↓12 ↑32 ↑226 Neutrophil ↓10 ↑11 ↑10 ↓15 Monocytes ↑16 ↑23 ↑17 ↑42 ↑76 ↓27



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Eosinophils ↓59 ↓65 ↓57 ↓55 ↓73 ↑79 Basophils ↑33 ↑33 ↑33 ↑50 LUC ↑114 ↑171 ↑50 ↑137 ↑117 ↓33

Day 224 represents the recovery data Summary:

• Test article-related effects included lower red blood cell counts, hemoglobin and hematocrit in the 5 mg/kg/day group males and the 10 mg/kg/day group males and females. Absolute reticulocyte counts were also lower in spite of lower red cell mass, which suggested a nonregenerative anemia and appeared to be reflective of a test article-related suppression of erythropoiesis. The study results showed a trend over time toward higher absolute neutrophil and monocyte counts (suggesting inflammation) and lower absolute lymphocyte and eosinophil counts, most prominent in the 10 mg/kg/day group males. Full or partial recovery was evident in hematologic parameters on study day 224.

Clinical Chemistry: unremarkable Urinalysis: unremarkable Ophthalmoscopy: unremarkable EKG: unremarkable Organ Weight: unremarkable Gross Pathology: Early deaths: Male #4411: Reddened areas of the gastrointestinal tract, thickened heart and

over 300 mL of red fluid in the thoracic cavity were observed macroscopically.

Male #4430: Reddened areas in the gastrointestinal tract, dark red and white discolorations on the lungs, lungs not fully collapsed and enlarged thyroid glands were noted.

Female #4454: Reddened areas in the gastrointestinal tract, dark red and white discolorations on the lungs, small and pale spleen, pale thyroid glands and approximately 100 mL of red fluid in the thoracic cavity were noted

Terminal deaths: The following summary table was excerpted from the submission



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Toxicologically Relevant Gross Necropsy Observations

Histopathology: Early death animals: Male #4411: Variable lymphoid depletion in lymphoid organs and parasitic mite-

related changes in the skin and mandibular lymph node were noted microscopically. Acute inflammation within the thoracic cavity was considered to be the cause of death.

Male #4430: moderate acute inflammation in the lungs morphologically consistent with a bacterial bronchopneumonia, which was considered to be the cause of death, and variable lymphoid depletion in the lymphoid organs were noted.

Female #4454: lymphoid depletion was evident in most sections of lymphoid tissue. Moderate bone marrow hypocellularity was noted. The cause of death was attributed to multifocal necrosis (with acute inflammation and bacteria) in the lung. Acute inflammation, necrosis and hemorrhage were noted in all 4 paws. There was also evidence of bacterial colonization of areas of skin with inflammation.

Terminal animals: Minimal-to-moderate acute or subacute inflammation was noted in the lungs of occasional dogs administered ≥ 2.5 mg/kg/day. Parasitic mites without inflammation were noted in one 2.5 mg/kg/day female. Lymphoid depletion within follicular centers and/or more generalized lymphoid depletion, and granulomatous inflammation with parasitic mites in regional lymph nodes were noted in animals at 5 and 10 mg/kg groups. Areas of inflammation occasionally included the



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presence of bacterial colonization. Squamous cell papilloma with basophilic intranuclear inclusion bodies in the skin was noted in 2 dogs (1 male and 1 female) administered 10 mg/kg/day of the drug. In the reproductive organs, prostate hypoplasia/atrophy was noted in 1 dog administered 5 mg/kg/day and 2 dogs administered 10 mg/kg/day. In the female dogs, the diestrus stage of the estrous cycle was noted in 1, 1, and 3 females administered 0.5, 5 and 10 mg/kg/day, respectively. Estrus was noted in 1 female administered 0.5 mg/kg/day and in 1 female administered 5 mg/kg/day. All control group females were in either anestrus or proestrus. A lack of regressing/residual corpora lutea and minimal mammary gland development in most females suggested that these dogs were either sexually immature or in their first estrous cycle.

At the recovery examination, pyogranulomatous inflammation in the skin, typically associated with mite and/or bacterial infestation, was noted in males and females that had been administered 5 or 10 mg/kg/day, with extension of inflammation into the mandibular lymph node. One control group female and 5 of 7 females administered INCB018424 were in diestrus.

Toxicokinetics: refer to PK section

STUDY SUMMARY: INCB018424 was administered to male and female dogs via oral gavage at doses of 0, 0.5, 2.5, 5, and 10 mg/kg/day daily for 6 months, followed by a 6-week recovery period. Systemic toxicity was observed at dosage levels of 5 and 10 mg/kg/day. Test article-related effects included 3 unscheduled deaths in the 10 mg/kg/day group animals with clinical findings of reddened areas, swelling and hair loss on the limbs, diarrhea and/or soft feces and clear and/or colored discharge from the eyes. The causes of death in all 3 dogs were attributed to inflammatory lesions in the lung/thoracic cavity (acute inflammation in both males and lung necrosis in the female). Generalized lymphoid depletion, granulomatous inflammation with parasitic mites, inflammatory cell infiltrates in the lymph nodes and acute and/or subacute inflammation of the lungs were noted microscopically in the ≥5 mg/kg/day group animals. Microscopic findings consisted of demodectic mange in one 2.5 mg/kg/day group animal.



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Study design: Male and female dogs were dosed daily via oral gavage for a total 52 weeks, followed by a 6-week recovery period (recovery groups). Note: male group at 6 mg/kg were terminated early (dosing phase week 29, dosing phase day 197)

OBSERVATIONS AND TIMES:

Mortality Two times daily Clinical examinations daily Detailed physical examinations weekly Body weights predose phase, weekly during the dosing

phase, on the first day of recovery phase, and weekly thereafter during the recovery phase.

Food consumption 1-week interval during the predose phase, weekly during the dosing phase, on the first day of recovery phase, and weekly thereafter during the recovery phase

Hematology and coagulation prior to randomization (study day -5) and on study days 27, 49, 86, 135 (hematology only), 174 and 224

Serum chemistry during the predose phase and once during dosing phase weeks 4, 12, 26, 39, and 52 (excluding Group 5 males) of the dosing phase additionally, a hematology sample was collected from Group 5 Male No. H49124 on dosing phase day 169 approximately 24 hours postdose (based on dosing time from dosing phase day 168) Blood and urine samples were collected prior to the early sacrifice of the surviving group 5 males on dosing phase day 197

Urinalysis once during the predose phase and once during dosing phase weeks 4, 12, 26, 39, and 52 (excluding Group 5 males) Blood and urine samples were collected prior to the early sacrifice of the surviving Group 5 males on Dosing Phase Day 197

Urine metabolite profiling Urine samples were collected once prior to dosing, on day 1, and during dosing phase weeks 4, 26, and 52 (excluding Group 5 males) of the dosing phase

Ophthalmoscopy during the predose phase, during week 52 of the dosing phase (excluding Group 5 males),



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and during week 6 of the recovery phase (excluding Group 5 males)

EKG Recorded once during the predose phase, during Week 52 of the dosing phase (excluding Group 5 males), and during week 6 of the recovery phase (excluding Group 5 males)

Gross pathology: All animals at sacrifice Organ weights: All animals at sacrifice Immunophenotype all animals during Week 26 (in conjunction

with the clinical pathology samples). Histopathology: All animals at sacrifice

Adequate Battery: yes (x), no ( ) Peer review: yes (x), no ( )

Toxicokinetics: day 1 and during weeks 4, 13, 26, and 52 (excluding Group 5 males) of the dosing phase within 1 hour of dosing and approximately 0.5, 1, 2, 4, 8, 12, and 24 hour postdose. Additionally, a sample was collected from Group 5 Male No. H49124 on Dosing Phase Day 169 approximately 24 hours postdose (based on dosing time from Dosing Phase Day 168). Surviving Group 5 males were bled on Dosing Phase Day 196 (Week 28) within 1 hour of dosing and approximately 0.5, 1, 2, 4, 8, 12, and 24 hours postdose. The 12 and 24 hour samples were collected from fasted dogs.

RESULTS: Mortality: four early deaths at 6 mg/kg/day. 1 female: on study day 197; moribund sacrifice 3 males: on study day 197; moribund sacrifice The moribund condition was attributed to inflammation of the skin/subcutis and footpad Clinical signs: Early death: cage sores, alopecia,black skin, broken skin, dry skin, red skin, scabs,

interdigital cysts, interdigital furunculosis, papillomatosis, and pododermatitis

Male #4411: reddened facial area, clear to colored discharge from the eyes, soft feces and or diarrhea were observed prior to death.

Male #4430: hypoactivity, prostration with labored respiration and swelling in the left hindlimb were noted. Other clinical observations noted prior to



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euthanasia included soft feces, diarrhea and clear and/or colored discharge from the eyes and warts on the left hindlimb.

Female #4454: Clinical observations prior to death included hypoactivity, increased respiration rate, labored respiration, diarrhea, swelling of the left and right forelimbs and hindlimbs and impaired use of the left and right hindlimbs.

Terminal sacrifices: unremarkable Bodyweight: unremarkable Food Consumption: unremarkable Hematology:



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Summary:

• Decreased red cell parameters including decreased red cell count (↓up to 17%), hemoglobin concentration (↓up to 22%) , and/or hematocrit percentage (↓up to 18%) was observed in both males and females, which was generally present in males from Day 24 through Day 197 of the dosing phase, and in females starting from Day 24 throughout the dosing phase. There was recovering trends in females, but not fully recovered by the end of the recovery period (day 40 of recovery phase);

• Decreased eosinophil counts were observed in females dogs at all dose levels from Day 83 (↓up to 45%), and males at doses of ≥ 3 mg/kg/day from Day 182 (↓up to 55%). These changes in eosinophil counts were reversed by Day 40 of the recovery phase;

• Lymphocyte counts of males given 6 mg/kg/day were decreased from Day 24 through Day 197 of the dosing phase (up to↓35%);


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• Other hematologic changes included an increase of neutrophil and monocyte counts (↑up to 40%) from Days 182 through 197 in males at 6 mg/kg/day, suggesting inflammation.

Notes: 1) Compensatory increase in reticulocytes was not observed; 2) Decreases in lymphocytes correlated with the microscopic observations of decreased lymphocytes in gut-associated lymphoid tissue, mandibular and mesenteric lymph nodes. Decreased lymphocyte counts also correlated with decreases in total T cells, CD4+ cells and CD8+ cells in males; 3) Increased neutrophil and monocyte counts of males from Days 182 through 197 correlated with the microscopic observation of cutaneous pyogranulomatous inflammation associated with demodex mange mites.

Clinical Chemistry: unremarkable Urinalysis: unremarkable Ophthalmoscopy: unremarkable EKG: unremarkable Organ Weight: unremarkable Gross Pathology: Abrasion, discoloration, scaling, thickening, and/or alopecia of the

skin/subcutis and footpad were observed in animals and females at doses ≥ 3 mg/kg, including early deaths. Lymph node enlargement (mandibular and other) was observed in two males given 6 mg/kg/day. Footpad abrasions were present at the end of the recovery phase, other findings were recovered.

Histopathology: Early death animals: The microscopic findings in the four moribund animals were

similar to those in the other animals given 6 mg/kg/day (see below), except minimal acute inflammation of the liver (Animal No. H49124), minimal hypocellularity (decreased neutrophil storage pool) of sternal bone marrow (Animal No. H49125), minimal acute inflammation of the mandibular lymph node (Animal No. H49128), and moderate acute fibrinous inflammation of the mesenteric lymph node (Animal No. H49162).

Note: Special histologic staining procedures for microorganisms (Periodic acid-Schiff and the Gram stains, Brown and Brenn and Brown and Hopps) did not reveal a cause for the inflammation in the mesenteric lymph node. Above findings in early dearth animals were considered secondary to the marked inflammation of the skin/subcutis and/or footpad present in these animals, which in the case of Animal Nos. H49124 and H49162 may have been accompanied by the systemic spread of opportunistic pathogens.



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All animals including early death animals and scheduled sacrificed animals: The following table is excerpted from the submission, the data have been verified by the reviewer.



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Recovery animals: The following table from the submission, the data have been verified by the reviewer.

Summary:

• A test article-related decrease in lymphocytes was noted microscopically in some organs of males and females given >3 mg/kg/day. The decrease in lymphocytes was most consistently evident in the gut-associated lymphoid tissue (GALT) of the ileum. Pyogranulomatous inflammation of the skin/subcutis (including the perianal skin present with the section of rectum) and footpad affected most of the animals given 6 mg/kg/day and some of those given 3 mg/kg/day. Decreased lymphocytes and pyogranulomatous inflammation of the skin/subcutis were reversible, but not fully recovered at the end of recovery phase.

Toxicokinetics: refer to PK section

STUDY SUMMARY: INCB018424 caused death when it was given to dogs daily at the dose of 6 mg/kg. Treatment lowered mean absolute lymphocytes, reduced eosinophil counts and decreased red cell parameters. Histopathology changes included decrease of lymphocytes in the gut-associated lymphoid tissue (GALT) of the ileum, in the cortex of the mandibular and mesenteric lymph nodes, and in the white pulp of the spleen. The development of generalized demodicosis, a secondary effect based of INCB018424, was considered adverse at dose levels of 3 and 6 mg/kg/day.



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Histopathology inventory

Study -519048 -519049 7456-271 Species Rat Dog Dog Adrenals X* X* X* Aorta X X X Bone Marrow smear

X X X

Bone (femur) X X X Brain X* X* X* Cecum X X X Cervix X X X Colon X X X Duodenum X X X Epididymis X X X Esophagus X X X Eye X X X Fallopian tube Gall bladder Gross lesions X X X Harderian gland X X X Heart X* X* X* Ileum X X X Injection site X Jejunum X X X Kidneys X* X* X* Lachrymal gland X X X Larynx X X Liver X* X* X* Lungs X* X* X* Lymph nodes, cervical

Lymph nodes mandibular

X X X

Lymph nodes, mesenteric

X X X

Mammary Gland Nasal cavity Optic nerves X Ovaries X X X oviducts X X X Pancreas X X X Parathyroid X Peripheral nerve Peyer’s patches X


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Pharynx X X Pituitary X* X* X* Prostate X X X Rectum X X X Salivary gland X X X Sciatic nerve X X X Seminal vesicles X X X Skeletal muscle X X X Skin X X X Spinal cord X X X Spleen X* X* X* Sternum X X Stomach X X X Teeth Testes X X X Thymus X* X* X* Thyroid X* X* X* Tongue X X X Trachea X X X Urinary bladder X X X Uterus X X X Vagina X X X Zymbal gland

X, histopathology performed *, organ weight obtained



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7 Genetic Toxicology

In Vitro Reverse Mutation Assay in Bacterial Cells (Ames) Study title: INCB018424: Salmonella plate incorporation mutagenicity assay

- reviewed by Dr. Barbara Hill under IND 77,455 Key findings: INCB01824 (free base) was not mutagenic in the Salmonella plate incorporation assay, under the conditions of this experiment. Study no.: AB22ZU.501.BTL Sponsor study no.: T06-01-electronic submission Conducting laboratory: Date of study initiation: 1-16-06 GLP compliance: No QA reports: No Drug, lot #, and % purity: INCB01824 (free base), Lot# 003 Vehicle: DMSO Methods Strains/species/cell line: Salmonella typhimurium strains TA98 and TA100 Concentrations used in definitive study: 1.5, 5.0, 15, 50, 150, 500, 1500 and 5000 μg/plate (±S9; S9 derived from Aroclor 1254 induced rat liver homogenate); 2 plates/dose Basis of concentration selection: No range finding study was performed. No appreciable toxicity or precipitate were noted at concentrations used in the definitive study. Negative controls: DMSO Positive controls: 1) TA 98 strain – 2-Nitrofluorene (-S9, 1 µg/plate) and 2-aminoanthracene (+S9, 1 µg/plate) 2) TA 100 strain – Sodium azide (-S9, 1 µg/plate) and 2-aminoanthracene (+S9, 1 µg/plate) Incubation and sampling times: Plates were incubated at 37 ± 2ºC for 2 days (S. typhimurinum) after treatment. Plates were counted for colony formation by hand after completion of the incubation period. Results Study validity:

A test article was considered to be positive if it produced at least a 2-fold increase in the mean revertants per plate for tester strains TA98 or TA100 and this increase in the mean number of revertants per plate had to be accompanied by a dose response to increasing concentrations of the test article.

Solvent control mean reversion frequencies fell within established ranges. Positive control results were appropriate showing a mean reversion frequency that was three times or more greater than the mean reversion frequency of the solvent control


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plates. The dose range selected for the definitive study was appropriate according to ICH guidelines. Study outcome:

The test article produced a negative response in the presence and absence of S-9 activation. All of the tester strains treated with the test article exhibited a mean reversion frequency that was similar to the corresponding solvent control. Study title Salmonella-Escherichia coli/mammalian-microsome reverse mutation

assay with a confirmatory assay - reviewed by Dr. Barbara Hill under IND 77,455

Key findings: INCB01824 phosphate was not mutagenic in the Ames test, under the conditions of this experiment. Study no.: 7456-188 Sponsor study no.: T06-08-01 Volume #, and page #: electronic submission Conducting laboratory: Date of study initiation: 7-24-06 GLP compliance: Yes QA reports: Yes Drug, lot #, and % purity: INCB01824 phosphate, Lot# 009 Vehicle: DMSO Methods Strains/species/cell line: Salmonella typhimurium strains TA98, TA100, TA1535 and TA1537; Escherichia coli strain WP2 uvrA Concentrations used in definitive study: 33.3, 100, 333, 1000, 2500 and 5000 µg/plate (±S9; S9 derived from Aroclor 1254 induced rat liver homogenate) in all tester strains; 3 plates/dose. A confirmatory assay was conducted using the same doses used in the definitive study.

Basis of concentration selection: The range finding study conducted with 6.67, 10.0, 33.3, 66.7,100, 333, 337, 1000, 3330 and 5000 µg/plate for tester strainsTA98 and WP2uvrA (± S9; S9 derived from Aroclor 1254 induced rat liver homogenate); 1 plate/dose. No precipitate was noted at any concentration. Very slight toxicity was noted at 5000 µg/plate in TA98 and no toxicity was noted at 5000 μg/plate in WP2uvrA. The maximum concentration used in the definitive study was 5000 μg/plate for tester strains based on the results of the range finding study.

Negative controls: DMSO Positive controls: Provided in following table


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Incubation and sampling times: Plates were incubated at 37 ± 2ºC for 2 days (S. typhimurinum) or 3 days (E. coli) after treatment. Plates were counted for colony formation by automated colony counter and/or by hand after completion of the incubation period. Results Study validity:

A test article was considered to be positive if it produced at least a 2-fold increase in the mean revertants per plate for tester strains TA98, TA100 or WP2uvrA or if it produced at least a 3-fold increase in mean revertants per plate for tester strains TA1535 or TA1537. This increase in the mean number of revertants per plate had to be accompanied by a concentration-response to increasing concentrations of the test article. Solvent control mean reversion frequencies fell within established ranges. Positive control results were appropriate showing a mean reversion frequency that was three times or more than the mean reversion frequency of the solvent control plates. The c range selected for the definitive study was appropriate according to ICH guidelines. Study outcome: The test article produced a negative response in the presence or absence of S-9 activation. All tester strains treated with the test article exhibited a mean reversion frequency that was similar to the corresponding solvent control.

In Vitro Assays in Mammalian Cells Study title INCB018424: Chromosomal aberrations in cultured human peripheral

blood lymphocytes reviewed by Dr. Barbara Hill under IND 77,455

Key findings: INCB01824 was negative in the human blood peripheral lymphocyte assay with or without S9 activation, under the conditions of this assay. Study no.: 7456-187 Sponsor study no.: T06-08-02 Volume #, and page #: electronic submission



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Conducting laboratory: Date of study initiation: 7-24-06 GLP compliance: Yes QA reports: Yes Drug, lot #, and % purity: INCB01824 phosphate, Lot# 009 Vehicle: DMSO Methods Strains/species/cell line: Human peripheral blood lymphocytes Doses used in definitive study:

In the initial chromosomal assay, concentrations of 5.43, 7.75, 11.1, 15.8, 22.6, 32.3, 46.1, 65.9, 94.1, 134, 192, 274, 392, 560 and 800 μg/ml INCB01824 phosphate were used for a 3-hr incubation (± S9); 2/concentration. Cultures treated with concentrations of 22.6, 32.3, 46.1 and 65.9 μg/ml INCB01824 phosphate without metabolic activation and 15.8, 22.6, 32.3 and 94.1 μg/ml INCB01824 phosphate with metabolic activation were analyzed for chromosomal aberrations.

In the confirmatory chromosomal assay, concentrations of 1.25, 2.50, 5.00, 10.0, 20.0, 40.0, 65.0, 80.0, 95.0 and 110 μg/ml INCB01824 phosphate were used for a 22-hr incubation (-S9) and concentrations of 5.0, 10.0, 20.0, 30.0, 40.0, 50.0, 65.0, 80.0, 95.0, 110, 125 and 150 μg/ml INCB01824 phosphate were used for a 3 hr incubation (+S9); 2/concentration. Cultures treated with concentrations of 10.0, 20.0, 50.0 and 65.0 μg/ml INCB01824 phosphate without metabolic activation and 10.0, 20.0, 50.0 and 95.0 μg/ml INCB01824 phosphate with metabolic activation were analyzed for chromosomal aberrations. Basis of concentration selection: The concentrations selected for analysis of chromosomal aberrations was based on the level of toxicity noted for the incubations. The high concentration was selected so that at least 50% reduction in the mitotic index relative to the solvent control was noted. For the initial chromosomal assay, the 65.9 μg/ml INCB01824 phosphate concentration exhibited a 56% mitotic index reduction for the 3-hour incubation (-S9) and the 94.1 μg/ml INCB01824 phosphate concentration exhibited a 51% mitotic index reduction for the 3-hour incubation (+S9). For the confirmatory chromosomal assay, the 65.0 μg/ml INCB01824 phosphate concentration exhibited a 55% mitotic index reduction for the 22-hour incubation (-S9) and the 95.0 μg/ml INCB01824 phosphate concentration exhibited a 54% mitotic index reduction for the 3-hour incubation (+S9). Negative controls: DMSO Positive controls: Mitomycin C (-S9): 0.75, 1.0 and 1.5 μg/ml for 3 hour exposure, 0.2, 0.3, and 0.4 μg/ml for 22 hour exposure. Cyclophosphamide (+S9): 20, 25 and 40 μg/ml for 3 hour exposure Incubation and sampling times:

Cell cultures were incubated with test article ± S9 for 3 hours and harvested 22 hours after treatment initiation. Cell cultures were incubated with test article –S9 for 22


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hours and then harvested for analysis. Slides were prepared from the harvested cultures and stained with 5% Giemsa solution for the analysis of mitotic index and chromosomal aberrations.

Results Study validity:

A test article was considered to be positive for inducing chromosomal aberrations if a significant increase (the difference was considered significant when p ≤0.05) in the number of cells with chromosomal aberrations is observed at one or more concentrations and a linear trend test demonstrates a concentration-response relationship. Solvent control mean reversion frequencies fell within established ranges. Positive control results were appropriate for this assay. The range of concentrations selected for the initial and confirmatory assays were appropriate according to ICH guidelines. Study outcome: No significant increase in cells with chromosomal aberrations was noted in the analyzed cultures.

In Vivo Clastogenicity Assay in Rodent (Micronucleus Assay) Study title INCB018424: In vivo rat bone marrow micronucleus assay

reviewed by Dr. Barbara Hill under IND 77,455 Key findings: INCB01824 was negative in the in vivo rat micronucleus assay, under the conditions of this experiment. Study no.: 7456-209 Sponsor study no.: T06-10-02 Volume #, and page #: electronic submission Conducting laboratory: Date of study initiation: 9-27-06 GLP compliance: Yes QA reports: Yes Drug, lot #, and % purity: INCB01824 phosphate, Lot# SS-IN2-175 Vehicle: 0.5% methylcellulose Methods Strains/species/cell line: Sprague-Dawley rats; 9 weeks; males: 266-304 g; females:196-233 g Doses used in definitive study: Single oral (gavage) doses of 0 (vehicle), 62.5, 125, 250 mg/kg INCB01824 phosphate; 10 ml/kg; 5/sex/dose/timepoint (an additional 3 high-dose males and 12 high-dose females were dosed as potential replacements)


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Basis of dose selection: Preliminary toxicity study was conducted with single oral

(gavage) doses of 250, 300 and 350 mg/kg INCB01824 phosphate, 10 ml/kg, 3/sex/dose. Mortality was noted in the mid- (1 male, 2 females) and high-dose groups (1 male, 2 females). Clinical signs of toxicity were noted in low-, mid- and high-dose groups which included squinted eyes, hypoactivity, ataxia, labored, irregular or audible respiration, lacrimation, body cold to touch, flattened posture, pale appearance and hunched posture. The high-dose was selected as 250 mg/kg based on the results of the preliminary toxicity study.

Negative controls: 0.5% methylcellulose Positive controls: Cyclophosphamide (60 mg/kg), oral (gavage) Incubation and sampling times: Single oral (gavage) doses of INCB01824 phosphate or cyclophosphamide were administered to rats. Bone marrow for analysis of nucleated cells was obtained from control and high-dose INCB01824 phosphate treated rats at 24 and 48 hours (5/sex/group/timepoint) after dose administration and at 24 hr after dose administration in low-dose, mid-dose, and positive control animals (5/sex/group).

Stained bone marrow slides were scored for micronucleus and the PCE (polychromatic erythrocytes) to NCE (normal chromatic erythrocytes) cell ratio. The micronucleus frequency (expressed as percent micronucleated cells) was determined by analyzing the number of micronucleated PCEs from at least 2000 PCEs per animal. The PCE:NCE ratio was determined by scoring the number of PCEs and NCEs observed while scoring at least 500 erythrocytes per animal. Results Study validity:

A test article was considered to be positive if a statistically significant increase in micronucleated PCEs for at least one dose level, and a statistically significant dose-related response were observed.

Solvent control mutant frequencies fell within established ranges. Positive control results were appropriate. Dose range selected for the definitive study was appropriate according to ICH guidelines.

Study outcome:

INCB01824 phosphate did not induce any statistically significant increases in micronucleated PCEs at any of the doses tested in this study. A high level of mortality was noted in high-dose females (16/25). No mortality was noted in low- or mid-dose animals or high-dose males. No clinical signs of toxicity were noted in low-dose animals. Clinical signs of toxicity noted in mid-dose animals included squinted eyes, ataxia, hypoactivity and/or reddened skin on the paws, ears and/or nose. Clinical signs of toxicity noted in high-dose animals included squinted eyes, ataxia, hypoactivity, reddened skin on the paws, ears, and/or nose, irregular or labored respiration, lacrimation, hyperactivity, convulsions, body that was cold to touch, and/or lateral recumbency. In addition, bone marrow toxicity was noted in the high dose group (20%



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decrease in PCE/NCE ratio compared to concurrent vehicle control). Due to the unexpected mortality in the high dose females, no slides were prepared for this group for the 48 hour timepoint.

8 Carcinogenicity The carcinogenicity study was reviewed by Dr. Miyun Tsai-Turton.

Study title: INCB018424: 26 week repeated dose oral carcinogenicity study in Tg.rasH2 mice

Adequacy of the carcinogenicity study and appropriateness of the test model: Yes. The Applicant followed eCAC recommendations. Key study findings:

Ruxolitinib was given to mice once daily at 0, 15, 45, and 125 mg/kg for 26 weeks via oral gavage. TK group was included in this study. Urethane (1000 mg/kg; three i.p. injection on Week 1) was used as positive control whereas 0.5% methylcellulose was used as vehicle control.

In main study animals, there were deaths in low-dose (1 M and 1 F) and mid-dose (3 M and 4 F) groups but not in high-dose group. In TK study animals, there was one death in high-dose (1 F) group.

No ruxolitinib related clinical sign was noted. There was a decrease in body weight gain in high-dose animals (11 % in male and 15% in female) but there were no signs of toxicity associated with ruxolitinib. Food consumption was decreased in treated males (all dose groups) and females (mid-dose/high-dose groups).

Decreased absolute and/or relative organ weights were noted (male: brain/kidneys/liver/testes and female: brain/kidneys/liver/heart/spleen). These were probably attributed to decreased body weights and no correlation to gross histological findings.

Neoplastic findings: ruxolitinib did not increase the incidence of neoplastic lesions. There were lesions/tumors in lungs (i.e. multiple adenomas and/or carcinoma in control/treated animals) and in spleen (i.e. hemangiosarcomas in treated animals), or in multiple organs such as skin/skeletal muscle (i.e. hemangiosarcomas/hemangiomas in control/treated animals, sarcoma in treated animals). Other tumors observed in control/treated animals included Harderian gland adenoma, liver adenoma, thyroid follicular adenoma, nasal adenocarcinoma, stomach papilloma, and thymus thymoma. All findings, however, either had low incidence rates or fell within historical control ranges established by .

Non-neoplastic findings: ruxolitinib increased the incidence/severity of non-neoplastic inflammatory lesions of the nasal cavity (i.e. minimal to moderate exudative inflammation).

Study no.: T09-02-03 Volume #, and page #: eCTD 4.2.3.4


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Conducting laboratory and location: Date of study initiation: Feb 17th (male) and Feb 19 (female) 2009 GLP compliance: Yes QA report: Yes Drug, lot #, and % purity: INCB018424, Lot No. BPR-07-101-B2-21, 99.6% purity CAC concurrence: Yes on SPA (special protocol assessment for dose selection and test model) Methods

Doses: 0, 15, 45, and 125 mg/kg/day INCB018424

Basis of dose selection: MTD Species/strain: hemizygous Tg.rasH2 mice Number/sex/group (main study): 25/sex/group Route, formulation, volume: oral gavage Frequency of dosing: daily for 182 to 183 consecutive days Satellite groups used for toxicokinetics or special groups: TK (44/sex/group) with CByB6F1 mice

Age: 8 weeks Animal housing: environmentally controlled room with temp of 69-75 °F and a relative humidity of 30-70% with a 12 hr light/12 hr D cycle

Restriction paradigm for dietary restriction studies: ad libitum access to drinking water and diet

Drug stability/homogeneity: stability (prepared in 0.5% methylcellulose at 0.005 to 1000 mg/ml was confirmed for 24 hrs at RT and 14 days under refrigerated conditions. Homogeneity was confirmed with the means of each location (top,


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middle, bottom) were within 7% of the overall mean and all replicates were with 5% of their respectively mean. The mean concentration results were within 10% of the theoretical concentration and all replicates were within 5% of their respective means.

Dual controls employed: Yes, vehicle control was 0.5% methylcellulose and the positive control was 1000 mg/kg urethane (via i.p. injection on Days 1, 3, and 5).

Interim sacrifices: Deviations from original study protocol: Yes but these were not expected to affect the outcome of the study.

Observation times

Mortality: twice daily Clinical signs: twice daily Body weights: once weekly from Day 1 to Week 13 and biweekly thereafter Food consumption: weekly Organ weight: organs (i.e. adrenal glands, brain, heart, kidneys, liver, spleen, and testes/ovaries) were collected and weighted on scheduled necropsy

Histopathology: Peer review: yes, organ tissues (see below) were collected on scheduled necropsy for microscopic evaluation.

Toxicokinetics: blood samples were collected from 3 animals/sex/dose/timepoint on Days 1-2 and 176-177 (predose, 0.5, 1, 2, 4, 8, and 24 hr post-dose for ruxolitinib-treated TK animals). Urine samples of designated TK animals (3 animals/cage) were collected during week 26 for metabolite identification.

Serum antibody analysis: blood collection from designated animals (10 M/10F predose, 10M/10F Group1 and surviving M/F Group 5 on Day 183) were collected via retroorbital sinus for possible future analysis (non-GLP).



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Results Mortality: mortalities (main study) were seen in 1 low-dose (LD) and 3 mid-dose (MD) male rats, 1 LD and 4 MD female rats (compared to 12 males and 12 females with urethane-treated animals). Mortality (TK study) was seen in 1 high-dose (HD) female. Clinical signs: there were no treatment-related effects on clinical observations. Clinical sings were either transient, with no dose-response, or appeared in a small number in treated animals (compared to clinical signs such as ataxia, decreased motor activity, labored breathing, hunched posture, hyperactivity, and hypothermia in urethane-treated animals). Body weights: there were decreases (but no dose-related effects) on body weights or body weight gains in LD and MD males. There was also 11% (males) and 15% (females) decreases in absolute body weight gain from Day 1 to Day 183 in HD group. Food consumption: There was a decrease in total food consumption from Day 1 to Day 183 in all treated males when compared to the vehicle control. There was also a significant decrease in total food consumption in MD and HD females when compared to the vehicle control. Organ weights: There were decreases in several absolute and relative organ weights (i.e. male: brain/kidneys/liver/testes and female: brain/kidneys/liver/heart/spleen). Such decreases (with no histological correlates) were considered to be secondary to decreased body weight.



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Gross pathology: There was no treatment-related effect on macroscopic findings in either sex (compared to the pulmonary and splenic lesions noted in urethane-treated animals) Histopathology: see appendix for detailed microscopic findings provided by the sponsor.

Non-neoplastic: Minimal to moderate exudative inflammation of the nasal cavity was observed.



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Nasal cavity Mostly minimal to mild inflammation of the submucosa of the nasal cavities was considered a background lesion. These were characterized by infiltration of mostly neutrophils and fewer lymphocytes in the submucosal glands and the interglandular spaces. The incidence of such lesion was increased slightly in the ruxolitinib treated mice in both sexes. Exudative inflammations of the nasal cavities were noted. These were characterized by accumulation of degenerate neutrophils, rare lymphocytes and macrophages, fibrin, and sloughed cells. This was only occasionally noted in the vehicle control mice. However, this incidence and severity of this inflammation were higher in the HD group but comparable in the LD and MD groups when compared to vehicle control in both sexes. Additional changes present in both lesion and inflammation include dilation of glands, presence of eosinophilic droplets, and hyperplasia of submucosal glands or lining epithelium (mostly minimal intensity). These changes were considered to be part of the inflammatory process. Adenocarcionoma of the nasal cavity was seen in 1 Group 3 male and 2 Group 3 females. This appeared to arise from the deep submucosal glands. The incidence of this nasal adenocarcinoma was low (with no dose relationship) and comparable with historical control range established by the


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Others A variety of other non-neoplastic lesions (i.e. minimal brain inflammation, mild aorta proliferation, minimal heart periarteritis, and minimal liver necrosis) were noted but all were considered to be background/spontaneous, incidental, and/or their incidences were comparable across the dose groups. Based on these findings, the nasal cavity was considered to be a target organ for non-neoplastic inflammatory changes.

Neoplastic: There was no significant increase in the incidence of neoplasia (i.e. lesions in lung, spleen, hemangiomas/hemangiosarcomas in spleen/other organs, sarcomas in multiple organs, non-vascular/sarcomas/pulmonary tumors in multiple organs).

Neoplastic lesions in lungs The incidence of single or multiple adenomas, carcinomas, and that of combined incidence of all pulmonary tumors in vehicle and ruxolitinib treated mice was comparable and fell within the historical control range established by There was significant increase in the incidence of pulmonary tumors in urethane-treated mice when compared to control mice, proving the validity of the test system.

Neoplastic lesions in spleen The incidence of splenic hemangiosarcomas was low and comparable between ruxolitinib treated dose groups and fell within the historical control range. There were no splenic hemangiosarcomas seen in the vehicle control mice of both sexes.


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There was significant increase in the incidence of splenic hemangiosarcomas in urethane-treated mice when compared to control mice, proving the validity of the test system.

Hemangiomas and hemangiosarcomas in multiple organs including spleen (under the general category of mesenchymal tumors) There was a non significant increase in the incidence of hemangiomas and hemangiosarcomas in multiple organs of Groups 4/5 males (i.e. spleen, skin, and skeletal muscle) and Groups 3/4 females (i.e. spleen and skin). However such increase was not dose-dependent. The incidence of all hemangiomas and hemangiosarcomas in multiple organs in vehicle as well as ruxolitinib treated dose groups fell within the historical control range established at

Sarcomas in multiple organs (under the general category of mesenchymal tumors) The incidence of these sarcomas was low and involved random organs and these were not present in control Group 1.


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Other tumors (non vascular, non-sarcomas, and non pulmonary) in multiple organs There was a non-dose dependent increase in the incidence of thymomas in Group 4 females. However, they were not seen in vehicle control and were not recorded in the historical control ranges. The incidence of all remaining tumors involved isolated organs and/or fell within the historical control ranges.

Based on these findings, the lung, spleen, skin, skeletal muscle were apparent to be target organs for neoplastic changes/lesions. However, ruxolitinib did not increase the incidence of these lesions. Most changes identified occurred with low incidences and with no dose-response, or fell within historical control ranges established by Toxicokinetics: TK analysis showed that there was minimal gender difference in CByB6F1 hybrid mice. The Cmax values did not always increase proportionally with dose and were similar between Day 1 and Day 176 in males, but decreased slightly (30-


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50%) in females on Day 176. The AUC values increased in a greater than dose proportionally and were generally similar between Day 1 and Day 176, except a slight decrease (39%) in HD females on Day 176. The t ½ values were higher in HD groups when compared to LD and MD groups.



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Methods Doses: 0, 10, 30, 60 mg/kg at dose volume of

Frequency of dosing: Dose volume: 10 mL/kg

Route of administration: oral gavage Formulation/Vehicle: 0.5% (w/v) methylcellulose (400 cps), in reverse

osmosis/deionized water Species/Strain: Crl:CD(SD) rats

Number/Sex/Group: 22/sex/dose Satellite groups: None

Study design: All males were dosed for at least 10 weeks and included at least 28 days prior to mating and throughout the mating phase. Females were dosed for at least 14 days prior to mating (premating phase), throughout the mating phase, and through Gestation Day 7 (GD 7). Treated males were paired with treated females during the mating phase. On GD 13, females were necropsied and the uterus of each was examined for the number of live and dead fetuses and resorptions. Ovaries were examined for the number of corpora lutea. All males were necropsied and evaluated for reproductive capacity.


none

Observations and Results Parameters and endpoints evaluated: mortality, clinical observations, body weight

and food consumption data, necropsy findings, cesarean section data, and reproductive organ weight data, estrous cycle, and sperm evaluations

Statistical evaluations: Levene’s test was done to test for variance homogeneity.

One-way analysis of variance [ANOVA] was used to analyze data. If the ANOVA was significant (p < 0.05), Dunnett’s t-test was used for control versus treated group comparisons. The sperm motility and total count was compared using the Kruskal-Wallis nonparametric ANOVA test. If a significant result was obtained at the 5% probability level, the Wilcoxon (Mann-Whitney U) test served as the post-hoc group comparison test (at the 5% one-tailed probability level).

Mortality Two females were sacrificed moribund (10 mg/kg on day 34 and 60 mg/kg on day 30. Note: As the moribundity of these animals did not display a dose-dependency, no clinical signs were noted with the exception of moribundity (pale appearance) and resorptions (red vaginal discharge), and there were no treatment related necropsy findings, these unscheduled deaths were not considered treatment-related.



156

Clinical Signs Early death: Prior to death, both of these animals were noted with clinical signs of a

pale appearance to the entire body, red vaginal discharge, red fluid in the pan, and/or both eyes pale.

Scheduled death (males and females): unremarkable

Body Weight Early death: unremarkable

Scheduled death: Male: Treatment-related decreases in mean body weight were observed in all

treatment groups. 10 mg/kg: ↓3 to 5%, compared to control from Day 35 to the end of study, which

also correlated with a decrease in mean body weight gain from Day 35 to the end of study.

30 mg/kg: ↓4-9%, beginning on Day 17, lasting to the end of study on Day 71, statistically significant compared to control from Day 38 until the end of the study.

60 mg/kg/day: ↓3-11%, beginning on Day 24, lasting to the end of study on Day 71; statistically significant compared to control from Day 38 until the end of the study.

Female: unremarkable Feed Consumption Male: 9% increase compared to control in mean food consumption was noted in the

60 mg/kg/day males from Days 0 to 28. Female: unremarkable Necropsy Male: There were no treatment-related effects on average organ weights or relative

organ weights, sperm count, sample, concentration and motility. Female: There were no treatment-related effects on the estrous cycling of females.

There were no treatment-related effects on pregnancy or copulation rates. Preimplantation loss was slightly increased in the 60 mg/kg/day group. This was mainly due to litter effects (Animal Nos. B55161 and B55163), and as the remaining 18 litters in the 60 mg/kg/day group were similar to controls. A treatment-related increase in postimplantation loss was noted in the 60 and 30 mg/kg/day groups. In the 60 mg/kg/day group, this increase was significant and was attributed to a 100% litter involvement in resorptions and an increase in the number of late resorptions compared to control.

Overall reproductive function data are presented in the following table (excerpted from the application, data have been verified by the reviewer).



158

Key Study Findings

• Maternal mortality was observed at 60 mg/kg; • Statistically significant decreased fetal weights (up to 9%) were observed at 60

mg/kg, the dose that resulted in mortality in 2 dams. • No maternal or embryo-fetal developmental effect was seen at 30 mg/kg/day

dose (NOAEL=30 mg/kg/day) • The dose of 60 mg/kg results in an AUC0-24 of 19 μM.hr which is 2 times the

human AUC0-24 at the highest dose of 25 mg, twice daily (50 mg/day). Methods

Doses: 15*, 30, 60 mg/kg/day *Due to a typographical error, the actual dose level was 14 mg/kg/day Dose justification: Dose selection was based on results from previous developmental dose-range finding toxicity study.

Frequency of dosing: once daily Dose volume: 10 mL/kg

Route of administration: oral gavage Formulation/Vehicle: 0.5% methylcellulose

Species/Strain: Female Crl:CD®(SD)IGS BR rats 201-236 g and 8-10 weeks at mating

Number/Sex/Group: 25 animals/group Study design: daily dosing from gestation days (GD) 7-20*,

Necropsy/cesarean on gestation day 22 *the day of confirmation of mating was considered GD1


none

Observations and Results Parameters and endpoints evaluated:

Reproductive parameters: the number and placement of uterine implantation

sites, the number of live and dead fetuses, the number of early or late resorptions, any abnormalities of the



159

placenta or embryonic sac, and the number of corpora lutea on each ovary.

Fetal examination: Each fetus (live or dead) was sexed, weighed, examined for external malformations/variations, visceral/skeletal malformations/variations).

Statistical evaluations: Quantitative data (body weights, body weight changes, food consumption, uterine weights, and uterine data) were analyzed using the Kolmogorov-Smirnov test for normality, the Levene Median test for equal variance, and by one-way Analysis of Variance (ANOVA). If either the normality or equal variance test failed, then the analysis was continued using the non-parametric Kruskal-Wallis ANOVA on rank transformed data. For parametric data, if the ANOVA indicated statistical significance among experimental groups then the Dunnett’s t-test was used to delineate which groups (if any) differed from the control. For non-parametric data, if the ANOVA indicated statistical significance among experimental groups then the Dunn’s test was used to delineate which groups (if any) differed from the control. The probability value of less than 0.05 (two-tailed) was used as the critical level of significance for all tests. The litter was used as the experimental unit.

TK: not conducted in this study. The Applicant used data from study T07-10-14 (GLP study, Oral Administration of INCB018424 via Gavage: Dose Range Study for Effects on Embryo-Fetal Development in Sprague Dawley Rats) to compare the plasma exposure (AUCs) in rats and humans. The following table is from the Applicant’s submission (Study# T07-10-14).

In life observations: Mortality: 2 early deaths at 60 mg/kg. DG18, GD22 (prior to the scheduled necropsy).



160

Clinical signs: Early death 1 death at GD18: there were no abnormal observations prior to death. 1 death at GD22: brown vaginal discharge was noted starting on GD 20. Scheduled death: unremarkable Body weight: Body weights and body weight changes for treated animals were

comparable to those of controls throughout the study. Food consumption: Overall, there were no differences between the groups in the

total amount of food consumed (GD 7-22).

Terminal and necropsy evaluations: Dams: Necropsy evaluation:

Early death: 1 death on GD 18: a discolored uterus with brown amoniotic fluid was noted at necropsy. 1 death on GD22: unremarkable

Scheduled death: unremarkable

Uterine and Ovarian parameters Changes in uterine weight: unremarkable Embryo-fetal data: Changes of mean fetal body weight Index Fetal weight (g) Percentage deviation from

control Group (mg/kg)

0 15 30 60 0 15 30 60

Male 5.6 5.8 5.6 5.3** - - - -5** Female 5.5 5.5 5.3 5.0** - - -4 -9** Male+ female 5.7 5.6 5.4 5.2** - - -5 -9** ** p≤0.01 Fetal morphological data: unremarkable Conclusion: Under the conditions of this study, INCB018424 was not teratogenic in rats; the maternal no-observed-adverse-effect level (NOAEL) and the fetal NOAEL are 30 mg/kg.



162

Methods

Doses: 10, 30, 60 mg/kg/day Dose justification: Dose selection was based on results from previous developmental dose-range finding toxicity study.

Frequency of dosing: once daily Dose volume: 10 mL/kg

Route of administration: oral gavage Formulation/Vehicle: 0.5% methylcellulose

Species/Strain: Female New Zealand white rabbits 3000-3873 g and 5-6 weeks at mating

Number/Sex/Group: 20 animals/group Satellite groups: 3 animals/group

Study design: daily dosing from gestation days (GD) 8-21*, Necropsy/cesarean on gestation day 30 *the day of confirmation of mating was considered GD1.

Deviation from study protocol: None Observations and Results Parameters and endpoints evaluated:

Reproductive parameters: the number and placement of uterine implantation sites, the

number of live and dead fetuses, the number of early or late resorptions, any abnormalities of the placenta or embryonic sac, and the number of corpora lutea on each ovary.

Fetal examination: Each fetus (live or dead) was sexed, weighed, examined for external malformations/variations, visceral/skeletal malformations/variations).

Statistical evaluations:

Quantitative data (body weights, body weight changes, food consumption, uterine weights, and uterine data) were analyzed using the Kolmogorov-Smirnov test for normality, the Levene Median test for equal variance, and by one-way Analysis of Variance (ANOVA). If either the normality or equal variance test failed, then the analysis was continued using the non-parametric Kruskal-Wallis ANOVA on ranktransformed



163

data. For parametric data, if the ANOVA indicated statistical significance among experimental groups then the Dunnett’s t-test was used to delineate which groups (if any) differed from the control. For non-parametric data, if the ANOVA indicated statistical significance among experimental groups then the Dunn’s test was used to delineate which groups (if any) differed from the control. The probability value of less than 0.05 (two-tailed) was used as the critical level of significance for all tests. The litter was used as the experimental unit.

TK: prior to dosing, and 0.5, 1, 2, 4, 8, and 24 hours after dosing on GD 8-9 and 21-22 Results In life observations: Mortality: one death at 60 mg/kg on GD21, and one became moribund on GD21. Clinical signs: Early death 1 death at GD21: there were no abnormal observations prior to death. 1 moribund at GD21 (animal#4359): moderate appetite loss, low posture, limping,

hypersensitivity to touch, and moderate languid behavior were noted Scheduled death: unremarkable Body weight: Body weights and body weight changes for treated animals were

comparable to those of controls throughout the study, except for the 1 moribund (animal #4359) which had notable weight loss from GD19.

Food consumption: Treatment with INCB018424 did not affect food consumption,

except animal No. 4359 had reduced food consumption from GD 9 onwards until becoming moribund on GD 21.

Terminal and necroscopic evaluations Doe: Necropsy evaluation:

Early death: 1 death on GD 21: failure of the lung to collapse. 1 moribund at GD21: red vaginal fluid was noted Scheduled death: unremarkable

Uterine and Ovarian parameters Changes of uterine weight: unremarkable



164

Other uterine or ovarian parameters: Doses (mg/kg/day) 0 10 30 60 Corpora lutea/rabbit 9.8 10.7 10.6 10.2 Implantation sites/rabbit 9.3 9.8 9.9 9.8 % pre-implantation loss/group (mean%)

5.5 7.9 6.7 4.8

Total post-implantation loss/group (mean%)

12 (6.8)

25 (11.7)

12 (6.9)

27 (15)

Total resorptions/rabbit 0.6 1.3 0.6 1.3 Late resorptions/rabbit 0.3 0.5 0.5 1.1* Early resorptions/rabbit 0.3 0.8 0.2 0.3 * p < 0.05 Note: Calculated values do not include animals which were not pregnant or did not survive to the scheduled termination. Summary: Treatment with INCB018424 at 60 mg/kg/day resulted in a significantly

higher number of late resorptions (1.1 per dam at 60 mg/kg/day vs. 0.3 per control dam).

Embryo-fetal data: Mean fetal body weight Index Fetal weight (g) Percentage deviation from

control Group (mg/kg)

0 10 30 60 0 10 30 60

Male 40 40 39 36* - - - -10* Female 39 40 39 35 - - - -10 Male+ female 39 40 38 36* - - - -8 ** p≤0.01

Summary: Treatment with INCB018424 resulted in lower fetal weights at 60 mg/kg. Fetal morphological data: unremarkable Toxicokinetics: The following tables are excerpted from the study report. Toxicokinetic parameters for 10 mg/kg were not calculated as plasma concentrations were below the quantitation limit (0.01 µM) in all but three samples.



165

Summary of INCB018424 toxicokinetics in pregnant rabbits given daily oral dose of 30

or 60 mg/kg INCB018424

Summary: • The mean plasma Cmax and AUC values for INCB018424 on GD 8 and 21

increased in a greater than dose proportional manner; • Cmax and AUC values on GD 21 were similar to those on GD 8; • Absorption and elimination were rapid • The AUC0-24 at 60 mg/kg dose (606 nM.hr) is 7% the human AUC at the human

dose of 50 mg/day Conclusion: Under the conditions of this study, INCB018424 was not teratogenic in rabbits; the maternal no-observed-adverse-effect level (NOAEL) and the fetal NOAEL are 30 mg/kg.



167

Parameters and endpoints evaluated: F0: Mortality, clinical signs, body weight during gestation and lactation, food

consumption during gestation, natural delivery and litter data and necropsy data F1: Maturation phases based on mortality, clinical signs, body weights, sexual

development, locomotor activity, learning ability, and estrous cycle data during the postweaning; Mortality, clinical signs, body weights, natural delivery and litter data and necropsy data on F1 females during the mating and gestation phases; Mortality, clinical signs, body weight, breeding behavior, and necropsy data on F1 males

F2: Survival and body weight on lactation day 1 (LD 1). Toxicokinetics: from F0 females via a jugular vein on GD 13 and LD 10 prior to dosing

and 1, 2, 4, 8, and 24 hours postdose as follows: Three animals in Group 5 (control) were bled prior to dosing and at 1 hour postdose. The first three animals in Groups 6, 7, and 8 were bled prior to dosing and at 2 and 8 hours postdose. The second three animals in Groups 6, 7, and 8 were bled at 1, 4, and 24 hours postdose.

Results: F0 dams In-life observations: Gestation

Mortality: Three F0 animals died at an unscheduled interval: one control female (B61091) was sacrificed on LD 1 after total litter death, one 5 mg/kg/day female (B61116) was sacrificed on GD 27 and was determined to be not pregnant, and one 30 mg/kg/day female (B61185) died as a result of a dosing-related accident on LD 8.

Note: The Applicant stated that above deaths are not considered treatment-related, the treatment is not considered to have adverse effect on survival. The reviewer agrees.

Clinical signs: unremarkable Body weight: unremarkable Food consumption: unremarkable

Natural Delivery, Litter, and Lactation

• No INCB018424-related effects on maternal survival, F1 survival, clinical signs, or maternal body weights were observed.

• Although the effect was slight, the mean gestation duration of 30 mg/kg/day animals was statistically significantly longer compared to control (22.1 versus 21.7 days, respectively) and greater than that seen in historical data (21.6 to 21.9 days; , 2005-2009).

• While not significant, the mean number of implantation sites and mean number of pups delivered at 30 mg/kg/day were also less than those found in historical data (11.76 versus 11.83-14.44 and 11.52 versus 11.62-13.21, respectively).


(b) (4)


168

• The mean number of live pups/litter with live pups at 30 mg/kg/day was also lower than control on LD 0 and precull LD 4 (11.12 versus 11.48 and 10.52 versus 11.38, respectively).

Toxicokinetics: report INCYTE DMB-08.25.1 Summary of INCB018424 Toxicokinetics in Female Sprague-Dawley Rats Given Daily

Oral Doses of INCB018424 from Gestation Day 6 through Lactation Day 20

Note: the above summary table is excerpted from the application Summary:

1) The plasma Cmax and AUC values generally increased in a greater than dose proportional manner;

2) The plasma Cmax and AUC values on GD 13 were generally comparable to those on LD 10;

3) Tmax and T1/2 are similar between DG13 and LD10; 4) AUC0-24 (μM*h) at 30 mg/kg: GD 13 = 4; LD 10 = 2.7.

Terminal and necropsy evaluations: unremarkable F1 offspring In-life observations: including the evaluation for F1 Postweaning (Maturation) and F1

Breeding, Gestation, Resting Phase Mortality: none Clinical signs: unremarkable

Body weight: With the exception of males on LD 7, the mean covariate-adjusted body weight of 30 mg/kg/day F1 pups was statistically lower, compared to control, on LDs 0, 4 (pre and postcull), 7, 14, and 21. The lower mean covariate body weights for both males and females on LD 7, 14, and 21 reflect the low body weight on LD 0 and lower early body weight gains rather than a continuing body weight effect.



170

mutagenicity. was present in the 6-month Tg.rasH2 carcinogenicity study at 0.17%. Refer to Appendix/Attachments section for the review of the submitted study reports.


(b) (4)


172

26-32 weeks: male: ↓ HD(↓31%-51%); female: ↓LD-HD(↓10%-42%); ↑at some timepoints and some animals

Ophthalmoscopy unremarkable unremarkable EKG unremarkable unremarkable Hematology

Male: ↓ Hbg: (↓4-7%), HD ↓hematocrit(↓3-7%), HD ↓WBC (↓5-32%), LD-HD Neut: -↓LD, LM(↓6-23%), -↑MH, HD(↑12-45%) ↓Lymphocyte (↓5-45%), LD-HD ↑Monocyte (↑10-20%), LD-HD except on week26 at HD ↓eosinophil (↓7-42%), LD-HD Except D26 at MH

(↑)and week 12 at HD (↑)

↓basophil (↓14-57%), LD-HD ↓LUC (↓12-63%), LD-HD All reversible

Female: ↓ Hbg: (↓2-4%), HD ↓WBC (↓6-39%), all doses ↑Neutrophil: (↑21-44)all

doses Except week 26, LD,

MH

(↓6-8%) ↓Lymphocyte: (↓9-

50%), LD-HD ↑Monocyte (↑10-20%), LD-HD except on week26

at HD ↓eosinophil (↓11-44%), LMD-HD ↓basophil (↓50-100%), LMD-HD ↓LUC (↓20-60%), LD-HD All reversible

Male: ↓ RBC: (↓4-27%), MHD, HD↓Hgb:(↓6-33%), MHD, HD ↓hematocrit: (↓7-30%), MHD, HD reticulocyte: ≤day135, ↓17-32%, MHD, HD, Day 174, ↑15-22%, MHD, HD ↑NEUT(↑5-71%), MHD, HD ↓LYM(↓11-33%), MHD, HD ↑MONO(↑15-102%), MHD, HD ↓eosinophil (↓17-64%), MHD, HD ↓basophil (↓25-50%), MHD, HD ↑LUC(↑20-100%), HD All reversible

Female: ↓ RBC: (↓6-14%), MHD, HD ↓Hgb:(↓6-19%), MHD, HD ↓hematocrit: (↓5-17%), MHD, HD reticulocyte: ≤day135, ↓5-30%, MHD, HD, Day 174, ↑10-47%, MHD, HD ↑NEUT(↑10-11%), HD ↑MONO(↑12-76%), MHD, HD ↓eosinophil (↓41-80%), MHD, HD ↑basophil (↑33-100%), MHD, HD ↑LUC(↑50-171%), MHD, HD All reversible, except for baso at MHD

Clinical chemistry

unremarkable unremarkable



175

consumption was observed at 60 mg/kg during GD 7-13.

• Decreased fetal weights were observed at 60 mg/kg;

Rabbit oral Daily (G8-21) 10, 30, 60 mg/kg (120, 360, 720 mg/m2) Embryofetal development • Mortality at 60

mg/kg; • Moderate appetite

loss, low posture, limping, hypersensitivity to touch, languid behavior, and decreases in body weight and food consumption were noted

• higher number of late resorptions at 60 mg/kg/day;

• lower fetal weights at 60 mg/kg

rat oral Daily (GD 6-LD 20

5, 15, 30 mg/kg (30, 90, 180 mg/m2) Prenatal and postnatal development • slightly prolonged

gestation period,



176

reduced number of implantation sites, and reduced number of pups delivered at 30 mg/kg/day F0 females;

• decreased mean initial body weights, mean body weight gain at 30 mg/kg/day F1 pups;

• The NOAEL for fertility indices and all maternal and embryofetal survival, growth, and development parameters is 30 mg/kg/day



178

Incubation and sampling times: 3 days Results Study validity: No. of replicates: 3

Counting method: not provided

Criteria for positive results: The positive controls induced a response equal to twice (or more) the negative control incidence, which is formally the criterion for a mutagenic activity of a compound. Exception is strain TA102 for which a factor of 1.5 over the concurrent negative control value is considered as positive result.

Selection of bacteria tester strains was adequate based upon Guideline for Industry: Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals (ICH S2A). Positive controls produced expected responses. Drug concentration selection was adequate based upon observed bacteriotoxicity or ICH S2 criteria.

Study outcome: Mean revertant counts in the presence of metabolic activation

Bacteria strain and number of revertant colonies per plate

Test article Conc. (μg/plate)

TA98 TA100 TA1535 TA97a TA102 15 32 136 15 255 447 50 34 127 18 249 596 150 32 132 22 210 586 500 31 125 13 174 458

1500 27 136 12 288 410

5000 31 133 17 243 * Positive control

2aminoanthracene Benzo(a)pyrene

2-nitrofluorene

1324 324

2073 228 3811 2176

DMSO - 46 170 16 244 465


(b) (4)


180

Strains: TA98, TA 100, TA1535, TA97a, and TA102 Concentrations used in definitive study:

TA98 and TA100: 15, 50, 150, 500, 1500, and 5000 μg/plate TA1535, TA97a and TA102, +S9: 15, 50, 150, 500, 1500, and 5000 μg/plate

Bacteriotoxicity was evident at 5000 μg/plate using strain TA102 –S9 and at ≥ 1500 μg/plate using strain TA102 +S9

Basis of concentration selection: bacteriotoxicity or ICH S2 criteria for the highest

concentration of 5000 μg/plate Metabolic activation system: Liver S9 mix from male rats, Aroclor 1254-pretreated,

25 µl/plate Negative controls: DMSO 100 μl/plate Positive controls:

Tester Strain Positive controls TA97a 2-aminoanthracene

9-Aminoacridine TA98 2-aminoanthracene

Benzo(a)pyrene 2-nitrofluorene

TA100 2-aminoanthracene sodium azide

TA102 2-aminoanthracene Mitomycin C





Selection of bacteria tester strains was adequate based upon Guideline for Industry: Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals (ICH S2A). Positive controls produced expected responses. Drug concentration selection was adequate based upon observed bacteriotoxicity or ICH S2 criteria.



181




TA98 TA100 TA1535 TA97a TA102 15 46 142 20 293 610 50 38 138 20 287 714 150 40 140 17 266 576 500 44 141 16 244 423

1500 36 127 13 242 *



2-nitrofluorene

1324 324

2073 228 3811 2176

DMSO - 46 170 16 244 465 Mean revertant counts in the absence of metabolic activation



TA98 TA100 TA1535 TA97a TA102 15 17 109 20 197 513 50 22 104 19 206 521 150 26 86 23 204 490 500 25 118 18 315 411

1500 29 98 19 312 461


2-nitrofluorene sodium azide

2aminoanthracene Mitomycin C

118 850

1261

4027

2520 DMSO - 25 114 20 463 463

* Bacteriotoxicity was evident at 5000 μg/plate using strain TA102 –S9 and at ≥ 1500 μg/plate using strain TA102 +S9

• The mean mutant numbers of negative control plates lay within the range of acceptable negative control values;

• The positive controls induced a response equal to twice (or more) the negative control incidence, which is formally the criterion for a mutagenic activity of a compound;

• There were no relevant increases in the number of revertants above the corresponding negative control values under any of the test conditions used after treatment with the test item.


(b) (4)

(b) (4)


183






Selection of bacteria tester strains was adequate based upon Guideline for Industry: Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals (ICH S2A). Positive controls produced expected responses. Drug concentration selection was adequate based upon observed bacteriotoxicity.




TA98 TA100 TA1535 TA97a TA102 15 35 130 12 240 552 50 31 122 20 243 640 150 35 160 9 226 595 500 30 151 20 303 458

1500 43 131 20 * *

5000 * * * * * Positive control


2-nitrofluorene

1324 324

2073 228 3811 2176




TA98 TA100 TA1535 TA97a TA102 15 27 131 22 233 555 50 27 126 29 236 546 150 22 102 28 301 620

500 25 129 17 244 543


(b) (4)

(b) (4)


185

Concentrations used in definitive study: TA98 and TA100, +S9: 15, 50, 150, 500, 1500, and 5000 μg/plate TA1535, TA97a and TA102, +S9: 15, 50, 150, 500, 1500, and 5000 μg/plate

The test article precipitated at 5000 μg/plate under all test conditions. In strain TA102 +S9 precipitation was also seen at 1500 μg/plate. Bacteriotoxicity was not evident in this study; however, precipitation prevented colony counting.

Basis of concentration selection: solubility Metabolic activation system: Liver S9 mix from male rats, Aroclor 1254-pretreated,











Selection of bacteria tester strains was adequate based upon Guideline for Industry: Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals (ICH S2A). Positive controls produced expected responses. Drug concentration selection was adequate based upon observed solubility.



186




TA98 TA100 TA1535 TA97a TA102 15 30 156 12 177 559 50 20 144 16 189 549 150 29 163 15 212 547 500 27 139 14 239 464

1500 34 141 23 247 410*

5000 * * * * * Positive control


2-nitrofluorene

1324 324

2073 228 3811 2176




TA98 TA100 TA1535 TA97a TA102 15 40 138 24 189 532 50 37 145 21 319 513 150 38 135 21 213 517 500 40 142 23 237 441

1500 35 149 33 253 512

5000 37* 169* 34* * * Positive control



118 850

1261

4027

2520 DMSO - 25 114 20 318 463

* The test article precipitated at 5000 μg/plate under all test conditions. In strain TA102 +S9 precipitation was also seen at 1500 μg/plate. Bacteriotoxicity was not evident in this study; however, precipitation prevented colony counting.



• Treatment with the test item did not increase the number of revertants above the corresponding negative control values under any of the test conditions used.


(b) (4)

(b) (4)


187

Comments and conclusions: The study was valid under the conditions of this study. is not mutagenic to test strains TA98, TA100, TA1535, TA97a and TA102

in the Ames reverse-mutation assay.

Study title: AMES test Key findings: Under the testing condition, is not mutagenic to test strains TA98, TA100, TA1535, TA97a and TA102 in the Ames reverse-mutation assay. Study no.: 1012578

Volume #, and page #: electronic submission, page 1-27 Conducting laboratory and location: Glowienke, PhD,

Basel/Switzerland site Date of study initiation: May 28, 2010 GLP compliance: no, following SOP QA reports: yes ( x ) no ( ) Drug, lot #, and % purity: Lot # 10026-120-2 Lot 3 Purity 99.1% Methods: plate incorporation Strains: TA98, TA 100, TA1535, TA97a, and TA102 Concentrations used in definitive study:

TA98 and TA100, +S9: 15, 50, 150, 500, 1500, and 5000 μg/plate TA1535, TA97a and TA102, +S9: 15, 50, 150, 500, 1500, and 5000 μg/plate

Basis of concentration selection: ICH S2 criteria for the highest concentration of 5000 μg/plate

Metabolic activation system: Liver S9 mix from male rats, Aroclor 1254-pretreated,






TA102 2-aminoanthracene


(b) (4)

(b) (4)

(b) (4)

(b) (4)


188

Mitomycin C TA1535 2-aminoanthracene

sodium azide Incubation and sampling times: 3 days Results Study validity: No. of replicates: 3



Selection of bacteria tester strains was adequate based upon Guideline for Industry: Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals (ICH S2A). Positive controls produced expected responses. Drug concentration selection was adequate based upon ICH S2 criteria. Study outcome: Mean revertant counts in the presence of metabolic activation



TA98 TA100 TA1535 TA97a TA102 15 77 135 30 2437 501 50 64 130 19 3511 529 150 77 180 17 3706 516 500 75 143 17 2340 492

1500 77 134 20 2701 463

5000 37 130 20 1892 363 Positive control


2-nitrofluorene

1324 324

2073 228 3811 2176




TA98 TA100 TA1535 TA97a TA102 15 29 120 22 769 356 50 28 125 23 986 418 150 22 116 14 1541 471 500 27 139 21 1041 440

1500 31 151 17 1269 450


(b) (4)

(b) (4)


190

QA reports: yes ( x ) no ( ) Drug, lot #, and % purity: Lot # 10026-120-1 Lot3 Purity: “not given”-stated in the study report Methods: plate incorporation Strains: TA98, TA 100, TA1535, TA97a, and TA102 Concentrations used in definitive study:

TA98 and TA100: 15, 50, 150, 500, 1500, and 5000 μg/plate TA1535, TA97a and TA102, +S9: 15, 50, 150, 500, 1500, and 5000 μg/plate

Bacteriotoxicity was evident at 5000 μg/plate using strain TA102 +/- S9. Basis of concentration selection: Bacteriotoxicity or ICH S2 criteria for the highest concentration of 5000 μg/plate Metabolic activation system: Liver S9 mix from male rats, Aroclor 1254-pretreated,










Criteria for positive results: The positive controls induced a response equal to twice (or more) the negative control incidence, which is formally the criterion for a mutagenic activity of a compound. Exception is strain


(b) (4)


191

TA102 for which a factor of 1.5 over the concurrent negative control value is considered as positive result.

Selection of bacteria tester strains was adequate based upon Guideline for Industry: Specific Aspects of Regulatory Genotoxicity Tests for Pharmaceuticals (ICH S2A). Positive controls produced expected responses. Drug concentration selection was adequate based upon bacteriotoxicity or ICH S2 criteria.




TA98 TA100 TA1535 TA97a TA102 15 21 128 13 172 543 50 22 154 16 209 578 150 23 153 17 211 582 500 27 157 21 246 441

1500 33 144 15 243 598

5000 26 143 17 313 616* Positive control


2-nitrofluorene

1324 324

2073 228 3811 2176




TA98 TA100 TA1535 TA97a TA102 15 22 122 9 204 529 50 19 127 14 243 633 150 28 130 19 244 628 500 28 122 17 308 551

1500 27 130 19 301 607




118 850

1261

4027

2520 DMSO - 25 114 20 318 463

* Bacteriotoxicity was evident at 5000 μg/plate using strain TA102 +/- S9.




(b) (4)

(b) (4)


192

• Treatment with the test item did not increase the number of revertants above the corresponding negative control values under any of the test conditions used.

Comments and conclusions: The study was valid under the conditions of this study.

is not mutagenic to test strains TA98, TA100, TA1535, TA97a and TA102 in the Ames reverse-mutation assay.

OVERALL CONCLUSIONS AND RECOMMENDATIONS Conclusions: The non-clinical studies adequately support the safety of ruxolitinib phosphate by oral route in myelofibrosis. See the EXECUTVE SUMMARY, Page 4, for an overall summary of nonclinical findings. Signatures: Reviewer Signature ___________________________________ Supervisor Signature_____________________________


(b) (4)


WEI CHEN10/26/2011



PHARMACOLOGY/TOXICOLOGY FILING CHECKLIST FOR NDA/BLA or Supplement

File name: 5_Pharmacology_Toxicology Filing Checklist for NDA_BLA or Supplement 010908

NDA/BLA Number: 202192 Applicant: Incyte Stamp Date: June 3, 2011,

Drug Name: ruxolitinib phosphate NDA/BLA Type: new commercial

On initial overview of the NDA/BLA application for filing:

Content Parameter

Yes

No

Comment 1 Is the pharmacology/toxicology section

organized in accord with current regulations and guidelines for format and content in a manner to allow substantive review to begin?

x

2

Is the pharmacology/toxicology section indexed and paginated in a manner allowing substantive review to begin?

x

*Appears acceptable.

3

Is the pharmacology/toxicology section legible so that substantive review can begin?

x

*Appears acceptable

4

Are all required (*) and requested IND studies (in accord with 505 b1 and b2 including referenced literature) completed and submitted (carcinogenicity, mutagenicity, teratogenicity, effects on fertility, juvenile studies, acute and repeat dose adult animal studies, animal ADME studies, safety pharmacology, etc)?

X

5

If the formulation to be marketed is different from the formulation used in the toxicology studies, have studies by the appropriate route been conducted with appropriate formulations? (For other than the oral route, some studies may be by routes different from the clinical route intentionally and by desire of the FDA).

n/a

6

Does the route of administration used in the animal studies appear to be the same as the intended human exposure route? If not, has the applicant submitted a rationale to justify the alternative route?

x

7 Has the applicant submitted a statement(s) that all of the pivotal pharm/tox studies have been performed in accordance with the GLP regulations (21 CFR 58) or an explanation for any significant deviations?

x

8 Has the applicant submitted all special studies/data requested by the Division during pre-submission discussions?

n/a


PHARMACOLOGY/TOXICOLOGY FILING CHECKLIST FOR NDA/BLA or Supplement

File name: 5_Pharmacology_Toxicology Filing Checklist for NDA_BLA or Supplement 010908

Content Parameter

Yes

No

Comment

9 Are the proposed labeling sections relative to pharmacology/toxicology appropriate (including human dose multiples expressed in either mg/m2 or comparative serum/plasma levels) and in accordance with 201.57?

x

10 Have any impurity – etc. issues been addressed? (New toxicity studies may not be needed.)

*x

11 Has the applicant addressed any abuse potential issues in the submission? n/a

12 If this NDA/BLA is to support a Rx to OTC switch, have all relevant studies been submitted?

n/a

* Issues generally identified during review. IS THE PHARMACOLOGY/TOXICOLOGY SECTION OF THE APPLICATION FILEABLE? ____yes____ If the NDA/BLA is not fileable from the pharmacology/toxicology perspective, state the reasons and provide comments to be sent to the Applicant. Please identify and list any potential review issues to be forwarded to the Applicant for the 74-day letter. Wei Chen, Ph.D 06/12/2012 Reviewing Pharmacologist Date Saber Haleh, Ph.D Team Leader/Supervisor Date



WEI CHEN06/14/2011



Documents

202192Orig1s000 - Food and Drug Administration · 2012. 1. 9. · NDA # 202,192 Reviewer: Wei Chen, Ph.D. 5 following a single oral dose of INCB018424. Administration of ruxolitinib